Artificial cell constructs for cellular manipulation

ABSTRACT

The present invention contemplates induction of immunological tolerance thereby providing permanent allograft acceptance. This method obviates the need for a lifelong regimen of immunosuppressive agents which can increase the risk of infection, autoimmunity, and cancer. Immunological tolerance is thought to be mediated by regulatory T lymphocytes (T reg  cells) with immunosuppressive capabilities. A therapeutically relevant platform comprising artificial constructs are contemplated comprising numerous soluble and surface bound T reg  cell stimulating factors that may induce tolerance following allograft transplantation. Such artificial constructs, being the size of a cell, have surface bound monoclonal antibodies specific to regulatory T-cell surface moieties and encapsulated soluble regulatory T-cell modulating factors.

FIELD OF THE INVENTION

The present invention is related to the field of inducing immunologicaltolerance by specifically manipulating immune-related cells. Suchimmunological tolerance may be induced by providing biomimeticartificial cells that bypass native immunological cells. For example,biomimetic artificial cell compositions are contemplated which comprisesoluble factors that activate specific immune-related blood cells,including, but not limited to, macrophages and/or monocytes. Suchfactors may comprise chemoattractant factors. These biomimeticartificial particles may further present a specific biomimetic surfacepattern that results in a targeted cell response such that the particlesrepresent artificial presenting cells.

BACKGROUND

The science of transplantation, now half of a century old, hasdramatically increased and improved the life of many individuals,including many children, with end stage diseases. Recent advancements inimmunosuppressive agents have substantially decreased rejection ofallografts over the past decade and a half in the United States.Hariharan et al., N Engl J Med 342, 605-612 (2000); and First, M. R.,Transplant Proc 34:1369-1371 (2002). However, to avoid both episodes ofacute rejection and the initiation of chronic rejection followingtransplantation, immunosuppressive drugs must be administered over theentire life of the organ recipient. Consequences of this long-termadministration are profound, including undesirable side effects,increasing the risk of infection, autoimmunity, heart disease, diabetes,and cancer. The chronic administration of these immunosuppressive drugs(especially when give systemically) lead to toxicity and significantside effects, thereby leaving the patient vulnerable to a variety ofdiseases and systemic organ failure.

The most desirable alternative to this extended state of vulnerabilitywould be to render the patient's immune system to effectively suppressimmune activation without systemic immunosuppression. In this case, nofurther immunosuppressant drug treatment would be necessary.Furthermore, the recipient's immune system would otherwise functionnormally, being capable of combating pathogens and malignant tumorcells. What is needed in the art are compositions and methods to inducepermanent immunological tolerance by stimulating immunosuppressiveregulatory cells.

SUMMARY OF THE INVENTION

The present invention is related to the field of inducing immunologicaltolerance by specifically manipulating immune-related cells. Suchimmunological tolerance may be induced by providing biomimeticartificial cells that bypass native immunological cells. For example,biomimetic artificial cell compositions are contemplated which comprisesoluble factors that activate specific immune-related blood cells,including, but not limited to, macrophages and/or monocytes. Suchfactors may comprise chemoattractant factors. These biomimeticartificial particles may further present a specific biomimetic surfacepattern that results in a targeted cell response such that the particlesrepresent artificial presenting cells.

In one embodiment, the present invention contemplates an artificialparticle comprising a plurality of soluble and surface bound regulatoryT-cell factors. In one embodiment, the artificial particle comprises anartificial presenting cell. In one embodiment, the factors compriseregulatory T cell stimulatory factors. In one embodiment, the factorscomprise regulatory T cell inducing factors. In one embodiment, thefactors comprise regulatory T cell chemoattractant factors. In oneembodiment, the soluble factors undergo controlled released. In oneembodiment, the controlled release is a short term release. In oneembodiment, the short term release is between 30 minutes-1 hour. In oneembodiment, the short term release is between 1 hour-3 hours. In oneembodiment, the short term release is between 3 hours-10 hours. In oneembodiment, the short term release is between 10 hours-24 hours. In oneembodiment, the controlled release is a long term release. In oneembodiment, the long term release is between 24 hours-36 hours. In oneembodiment, the long term release is between 3 days-7 days. In oneembodiment, the long term release is between 7 days-1 month. In oneembodiment, the long term release is between 1 month-6 months. In oneembodiment, the long term release is between 6 months-1 year. In oneembodiment, the long term release is at least one year. In oneembodiment, the surface bound factors comprises a biomimetic surfacepattern. In one embodiment, the biomimetic surface pattern comprises animmunological synapse. In one embodiment, the artificial presenting celltake a shape selected from the group consisting of a sphere, a square, arectangle, a triangle, a trapezoid, a hexagon, an octagon, or atetrahedron.

In one embodiment, the present invention contemplates an artificialparticle comprising at least one soluble T_(reg) cell factor and atleast one surface bound T_(reg) cell factor, wherein said surface boundfactor is displayed in a non-random pattern. In one embodiment, theartificial particle comprises an artificial presenting cell. In oneembodiment, the at least one soluble or surface bound Treg cell factorcomprises a regulatory T cell stimulatory factor. In one embodiment, theat least one soluble or surface bound Treg cell factor comprises aregulatory T cell inducing factor. In one embodiment, the at least onesoluble or surface bound Treg cell factor comprises a regulatory T cellchemoattractant factor. In one embodiment, the artificial presentingcell further comprises a functionalized polymer, wherein said polymerencapsulates the soluble factor. In one embodiment, the artificialpresenting cell further comprises compartments, wherein saidcompartments comprise the at least one soluble factor. In oneembodiment, the artificial presenting cell is biodegradable such thatthe soluble factor undergoes controlled release. In one embodiment, thesoluble factor comprises CCL22. In one embodiment, the soluble factorcomprises IL2. In one embodiment, the soluble factor comprises TGF-β. Inone embodiment, the polymer comprises poly-lactic acid-co-glycolic acid(PLGA). In one embodiment, the functionalized polymer non-covalentlybonds the soluble factor. In one embodiment, the at least one surfacebound T_(reg) stimulating factor forms a biomimetic surface pattern. Inone embodiment, the biomimetic surface pattern comprises animmunological synapse. In one embodiment, the synapse comprises a cSMACregion, displayed on an apical portion of the artificial presentingcell. In one embodiment, the synapse comprises a pSMAC region displayedon a tropical region of the artificial presenting cell. In oneembodiment, the synapse comprises a dSMAC region displayed on anequatorial portion of the artificial presenting cell. In one embodiment,the cSMAC region comprises CD3 antibodies and/or CD28 antibodies. In oneembodiment, the pSMAC region comprises at least one adhesion molecule.In one embodiment, the dSMAC region comprises a CD45 monoclonalantibody. In one embodiment, the functionalized polymer covalently bondsthe surface bound regulatory T cell stimulatory factor.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) an emulsified microparticle comprising atleast one soluble T_(reg) cell factor and a functionalized polymer; andii) at least one surface bound T_(reg) cell factor, wherein the surfacebound factor is capable of attaching to the polymer; b) attaching afirst surface bound factor to said polymer, wherein an apical region iscreated; c) attaching a second surface bound factor to said polymer,wherein a tropical region is created; and d) attaching a third surfacebound factor to said polymer, wherein an equatorial region is created.In one embodiment, the microparticle comprises an artificial presentingcell. In one embodiment, the at least one soluble or surface bound Tregcell factor comprises a regulatory T cell stimulatory factor. In oneembodiment, the at least one soluble or surface bound Treg cell factorcomprises a regulatory T cell inducing factor. In one embodiment, the atleast one soluble or surface bound Treg cell factor comprises aregulatory T cell chemoattractant factor. In one embodiment, themicroparticle is biodegradable such that the soluble factor undergoescontrolled release. In one embodiment, the soluble factor comprisesCCL22. In one embodiment, the soluble factor comprises IL2. In oneembodiment, the soluble factor comprises TGF-β. In one embodiment, theapical, tropical, and equatorial regions comprise a biomimetic pattern.In one embodiment, the biomimetic pattern comprises an immunologicalsynapse. In one embodiment, the first surface bound factor is selectedfrom the group consisting of a CD3 antibody and a CD28 antibody. In oneembodiment, the second surface bound factor comprises an adhesionprotein. In one embodiment, the third surface bound factor comprises aCD45 antibody. Alternatively, the microparticle may further comprise aCD3 ligand (i.e., MHC) and/or a CD28 ligand.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a polymer capable of undergoingemulsification, ii) at least one soluble T_(reg) cell factor, and iii)at least one surface bound T_(reg) cell factor; and b) emulsifying thepolymer and the soluble factor such that a biodegradable microparticleis created, such that the soluble factor undergoes controlled release.In one embodiment, the microparticle comprises an artificial presentingcell. In one embodiment, the at least one soluble or surface bound Tregcell factor comprises a regulatory T cell stimulatory factor. In oneembodiment, the at least one soluble or surface bound Treg cell factorcomprises a regulatory T cell inducing factor. In one embodiment, the atleast one soluble or surface bound Treg cell factor comprises aregulatory T cell chemoattractant factor. In one embodiment, theemulsifying may be selected from the group including, but not limitedto, precipitation, emulsions, melt casting, spray drying,crystallization, shearing, or milling. In one embodiment, themicroparticle is porous. In one embodiment, the microparticle comprisescompartment. In one embodiment, the method further comprises step (c)immobilizing a first surface bound stimulatory factor in an apicalregion of the microparticle. In one embodiment, the method furthercomprises step (d) immobilizing a second surface bound stimulatoryfactor in a tropical region of the microparticle. In one embodiment, themethod further comprises step (e) immobilizing a third surfacestimulatory factor in an equatorial region of the microparticle. In oneembodiment, the polymer comprises poly-lactic acid-co-glycolic acid(PLGA). In one embodiment, the soluble stimulatory factor is selectedfrom the group consisting of CCL22, IL2, and TGF-β. In one embodiment,the first surface bound factor is selected from the group consisting ofCD3 antibody and CD28 antibody. In one embodiment, the second surfacebound factor comprises an adhesion molecule. In one embodiment, thethird surface bound factor comprises a CD45 antibody. In one embodiment,the at least one surface bound signaling factor is randomly displayed onthe microparticle.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a patient having received a tissuetransplant, wherein said tissue is capable of immunolgical tolerance;ii) a artificial antigen presenting cell comprising at least one solubleT_(reg) cell factor and at least one surface bound T_(reg) cell factor;and b) administering the artificial antigen presenting cell to thepatient under conditions such that an immunological tolerant state isinduced in the tissue. In one embodiment, the at least one soluble orsurface bound Treg cell factor comprises a regulatory T cell stimulatoryfactor. In one embodiment, the at least one soluble or surface boundTreg cell factor comprises a regulatory T cell inducing factor. In oneembodiment, the at least one soluble or surface bound Treg cell factorcomprises a regulatory T cell chemoattractant factor. In one embodiment,the artificial antigen presenting cell is porous. In one embodiment, theimmunosuppression is not antigen-specific. In one embodiment, theartificial antigen presenting cell comprises compartments. In oneembodiment, the compartments comprise the soluble factor. In oneembodiment, the artificial antigen presenting cell is biodegradable,wherein the soluble factor undergoes controlled release. In oneembodiment, the controlled release is a short term release. In oneembodiment, the short term release is between 30 minutes-1 hour. In oneembodiment, the short term release is between 1 hour-3 hours. In oneembodiment, the short term release is between 3 hours-10 hours. In oneembodiment, the short term release is between 10 hours-24 hours. In oneembodiment, the controlled release is a long term release. In oneembodiment, the long term release is between 24 hours-36 hours. In oneembodiment, the long term release is between 3 days-7 days. In oneembodiment, the long term release is between 7 days-1 month. In oneembodiment, the long term release is between 1 month-6 months. In oneembodiment, the long term release is between 6 months-1 year. In oneembodiment, the long term release is at least one year. In oneembodiment, the method further comprises step (c) inducing a tissuetolerance by the immunosuppressive state. In one embodiment, the tissuetolerance is permanent. In one embodiment, the soluble stimulatoryfactor is selected from the group consisting of CCL22, IL2, and TGF-β.In one embodiment, the surface bound factors are selected from the groupconsisting of CD3 antibody, CD28 antibody, an adhesion molecule, and aCD45 antibody. Alternatively, the microparticle may further comprise aCD3 ligand (i.e., MHC) and/or a CD28 ligand.

In one embodiment, the present invention contemplates a kit comprising acontainer comprising at least one artificial particle suspended in apharmaceutically acceptable vehicle. In one embodiment, the artificialparticle comprises an artificial presenting cell. In one embodiment, thekit further comprises instructions for using the artificial particle toinduce immunosuppression in a patient. In one embodiment, the artificialparticle comprises at least one encapsulated soluble T_(reg) factor. Inone embodiment, the at least one soluble or surface bound Treg cellfactor comprises a regulatory T cell stimulatory factor. In oneembodiment, the at least one soluble or surface bound Treg cell factorcomprises a regulatory T cell inducing factor. In one embodiment, the atleast one soluble or surface bound Treg cell factor comprises aregulatory T cell chemoattractant factor. In one embodiment, the solublefactor is selected from the group consisting of CCL22, IL2 or TGF-β.

In one embodiment, the present invention contemplates an artificialosteoblast cell comprising a plurality of soluble and surface boundosteoclast signaling factors. In one embodiment, the soluble signalingfactors are controlled released. In one embodiment, the solublesignaling factor comprises MCSF. In one embodiment, the surface boundsignaling factor comprises RANK-L, OSCAR-L, and/or ODF. In oneembodiment, the controlled release is a short term release. In oneembodiment, the short term release is between 30 minutes-1 hour. In oneembodiment, the short term release is between 1 hour-3 hours. In oneembodiment, the short term release is between 3 hours-10 hours. In oneembodiment, the short term release is between 10 hours-24 hours. In oneembodiment, the controlled release is a long term release. In oneembodiment, the long term release is between 24 hours-36 hours. In oneembodiment, the long term release is between 3 days-7 days. In oneembodiment, the long term release is between 7 days-1 month. In oneembodiment, the long term release is between 1 month-6 months. In oneembodiment, the long term release is between 6 months-1 year. In oneembodiment, the long term release is at least one year. In oneembodiment, the surface bound signaling factors comprises a biomimeticsurface pattern. In one embodiment, the biomimetic surface patterncomprises an osteological synapse. In one embodiment, the artificialosteoblast cell take a shape selected from the group consisting of asphere, a square, a rectangle, a triangle, a trapezoid, a hexagon, anoctagon, or a tetrahedron.

In one embodiment, the present invention contemplates an artificialosteoblast cell comprising at least one soluble osteoclast cellsignaling factor and at least one surface bound osteoclast cellsignaling factor. In one embodiment, the artificial osteoblast cellfurther comprises a functionalized polymer, wherein said polymerencapsulates the soluble factor. In one embodiment, the artificialosteoblast cell further comprises compartments, wherein saidcompartments comprise the soluble signaling factor. In one embodiment,the artificial osteoblast cell is biodegradable such that the solublefactor undergoes controlled release. In one embodiment, the solublefactor comprises MCSF. In one embodiment, the polymer comprisespoly-lactic acid-co-glycolic acid (PLGA). In one embodiment, thefunctionalized polymer non-covalently bonds the soluble factor. In oneembodiment, the at least one surface bound osteoclast signaling factorforms a biomimetic surface pattern. In one embodiment, the biomimeticsurface pattern comprises an osteological synapse. In one embodiment,the surface bound osteoclast signaling factor comprises RANK-L, OSCAR-L,and/or ODF. In one embodiment, the functionalized polymer covalentlybonds the surface bound osteoclast cell stimulatory factor.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) an emulsified microparticle comprising atleast one soluble osteoclast cell signaling factor and a functionalizedpolymer; and ii) at least one surface bound osteoclast cell signalingfactor, wherein the surface bound signaling factor is capable ofattaching (i.e., for example, by conjugating and/or adsorbing) to thepolymer; b) attaching a first surface bound factor to said polymer,wherein a first region is created; c) attaching a second surface boundfactor to said polymer, wherein a second region is created; and d)attaching a third surface bound factor to said polymer, wherein a thirdregion is created. In one embodiment, the microparticle is biodegradablesuch that the soluble factor undergoes controlled release. In oneembodiment, the at least one soluble signaling factor comprises MSCF. Inone embodiment, the first, second, and third regions comprise abiomimetic pattern. In one embodiment, the biomimetic pattern comprisesan osteological synapse. In one embodiment, the first surface boundsignaling factor comprises RANK-L, OSCAR-L, and/or ODF.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a polymer capable of undergoingemulsification, ii) at least one soluble osteoclast cell signalingfactor, and iii) at least one surface bound osteoclast cell signalingfactor; and b) emulsifying the polymer and the soluble signaling factorsuch that a biodegradable microparticle is created, such that thesoluble factor undergoes controlled release. In one embodiment, themicroparticle is porous. In one embodiment, the microparticle comprisescompartments. In one embodiment, the emulsifying may be selected fromthe group including, but not limited to, precipitation, emulsions, meltcasting, spray drying, crystallization, shearing, or milling. In oneembodiment, the method further comprises step (c) immobilizing a firstsurface bound signaling factor in an apical region of the microparticle.In one embodiment, the method further comprises step (d) immobilizing asecond surface bound signaling factor in a tropical region of themicroparticle. In one embodiment, the method further comprises step (e)immobilizing a third surface signaling factor in an equatorial region ofthe microparticle. In one embodiment, the polymer comprises poly-lacticacid-co-glycolic acid (PLGA). In one embodiment, the soluble signalingfactor comprises MCSF. In one embodiment, the at least one surface boundsignaling factor comprises RANK-L, OSCAR-L, and/or ODF. In oneembodiment, the at least one surface bound signaling factor is randomlydisplayed on the microparticle.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a patient, wherein said patient is in needof bone healing; ii) an artificial osteoblast cell comprising at leastone soluble osteoclast cell signaling factor and at least one surfacebound osteoclast cell signaling factor; and b) administering theartificial osteoblast cell to the patient under conditions such that thebone healing is induced in the patient. In one embodiment, the patienthas under gone bone surgery. In one embodiment, the patient comprises abone injury. In one embodiment, the patient comprises a bone disease. Inone embodiment, the artificial osteoblast cell is porous. In oneembodiment, the artificial osteoblast cell comprises compartments. Inone embodiment, the compartments comprise the soluble signaling factor.In one embodiment, the artificial osteoblast cell is biodegradable,wherein the soluble factor undergoes controlled release. In oneembodiment, the controlled release is a short term release. In oneembodiment, the short term release is between 30 minutes-1 hour. In oneembodiment, the short term release is between 1 hour-3 hours. In oneembodiment, the short term release is between 3 hours-10 hours. In oneembodiment, the short term release is between 10 hours-24 hours. In oneembodiment, the controlled release is a long term release. In oneembodiment, the long term release is between 24 hours-36 hours. In oneembodiment, the long term release is between 3 days-7 days. In oneembodiment, the long term release is between 7 days-1 month. In oneembodiment, the long term release is between 1 month-6 months. In oneembodiment, the long term release is between 6 months-1 year. In oneembodiment, the long term release is at least one year. In oneembodiment, the bone surgery comprises bone replacement including, butnot limited to, hip replacement or knee replacement. In one embodiment,the bone surgery comprises bone reconstruction. In one embodiment, thebone surgery comprises bone fracture repair. In one embodiment, thesoluble signaling factor comprises MCSF. In one embodiment, the surfacebound signaling factor comprises RANK-L, OSCAR-L, and/or ODF.

In one embodiment, the present invention contemplates a kit comprising acontainer comprising at least one artificial osteoblast cell suspendedin a pharmaceutically acceptable vehicle. In one embodiment, the kitfurther comprises instructions for using the artificial osteoblast cellto induce bone healing in a patient. In one embodiment, the artificialosteoblast cell comprises at least one encapsulated soluble osteoclastsignaling factor. In one embodiment, the soluble signaling factorcomprises MCSF.

In one embodiment, the present invention contemplates an artificialosteoclast cell comprising a plurality of soluble and surface boundosteoblast signaling factors. In one embodiment, the soluble signalingfactors are controllably released. In one embodiment, the solublesignaling factor comprises MIM1. In one embodiment, the surface boundsignaling factor comprises EphrinB2 and/or TGF-β. In one embodiment, thecontrolled release is a short term release. In one embodiment, the shortterm release is between 30 minutes-1 hour. In one embodiment, the shortterm release is between 1 hour-3 hours. In one embodiment, the shortterm release is between 3 hours-10 hours. In one embodiment, the shortterm release is between 10 hours-24 hours. In one embodiment, thecontrolled release is a long term release. In one embodiment, the longterm release is between 24 hours-36 hours. In one embodiment, the longterm release is between 3 days-7 days. In one embodiment, the long termrelease is between 7 days-1 month. In one embodiment, the long termrelease is between 1 month-6 months. In one embodiment, the long termrelease is between 6 months-1 year. In one embodiment, the long termrelease is at least one year. In one embodiment, the surface boundsignaling factors comprises a biomimetic surface pattern. In oneembodiment, the biomimetic surface pattern comprises an osteologicalsynapse. In one embodiment, the artificial osteoblast cell take a shapeselected from the group consisting of a sphere, a square, a rectangle, atriangle, a trapezoid, a hexagon, an octagon, or a tetrahedron.

In one embodiment, the present invention contemplates an artificialosteoclast cell comprising at least one soluble osteoblast cellsignaling factor and at least one surface bound osteoblast cellsignaling factor. In one embodiment, the artificial osteoclast cellfurther comprises a functionalized polymer, wherein said polymerencapsulates the soluble factor. In one embodiment, the functionalizedpolymer comprises a surface polymer. In one embodiment, the artificialosteoclast cell further comprises compartments, wherein saidcompartments comprise the soluble signaling factor. In one embodiment,the artificial osteoclast cell is biodegradable such that the solublefactor undergoes controlled release. In one embodiment, the solublefactor comprises MIM1. In one embodiment, the polymer comprisespoly-lactic acid-co-glycolic acid (PLGA). In one embodiment, thefunctionalized polymer non-covalently bonds the soluble factor. In oneembodiment, the at least one surface bound osteoblast signaling factorforms a biomimetic surface pattern. In one embodiment, the biomimeticsurface pattern comprises an osteological synapse. In one embodiment,the surface bound osteoblast signaling factor comprises EphrinB2 and/orTGF-β. In one embodiment, the functionalized polymer covalently bondsthe surface bound osteoclast cell stimulatory factor.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) an emulsified microparticle comprising atleast one soluble osteoblast cell signaling factor and a functionalizedpolymer; and ii) at least one surface bound osteoblast cell signalingfactor, wherein the surface bound signaling factor is capable ofattaching to the polymer; b) attaching a first surface bound factor tosaid polymer, wherein an apical region is created; c) attaching a secondsurface bound factor to said polymer, wherein a tropical region iscreated; and d) attaching a third surface bound factor to said polymer,wherein an equatorial region is created. In one embodiment, themicroparticle is biodegradable such that the soluble factor undergoescontrolled release. In one embodiment, the at least one solublesignaling factor comprises MIM1. In one embodiment, the apical,tropical, and equatorial regions comprise a biomimetic pattern. In oneembodiment, the biomimetic pattern comprises an osteological synapse. Inone embodiment, the first surface bound signaling factor comprisesEphrinB2 and/or TGF-β.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a polymer capable of undergoingemulsification, ii) at least one soluble osteoblast cell signalingfactor, and iii) at least one surface bound osteoblast cell signalingfactor; and b) emulsifying the polymer and the soluble signaling factorsuch that a porous biodegradable microparticle is created, such that thesoluble factor undergoes controlled release. In one embodiment, theemulsifying may be selected from the group including, but not limitedto, precipitation, emulsions, melt casting, spray drying,crystallization, shearing, or milling. In one embodiment, the methodfurther comprises step (c) immobilizing a first surface bound signalingfactor in an apical region of the microparticle. In one embodiment, themethod further comprises step (d) immobilizing a second surface boundsignaling factor in a tropical region of the microparticle. In oneembodiment, the method further comprises step (e) immobilizing a thirdsurface signaling factor in an equatorial region of the microparticle.In one embodiment, the polymer comprises poly-lactic acid-co-glycolicacid (PLGA). In one embodiment, the soluble signaling factor comprisesMIM1. In one embodiment, the at least one surface bound signaling factorcomprises EphrinB2 and/or TGF-β.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a patient having undergone bone surgery,wherein said patient is in need of bone healing; ii) a porous artificialosteoclast cell comprising at least one soluble osteoblast cellsignaling factor and at least one surface bound osteoblast cellsignaling factor; and b) administering the artificial osteoclast cell tothe patient under conditions such that a bone healing is induced in thepatient. In one embodiment, the porous artificial osteoclast cellcomprises compartments. In one embodiment, the compartments comprise thesoluble signaling factor. In one embodiment, the artificial osteoclastcell is biodegradable, wherein the soluble factor undergoes controlledrelease. In one embodiment, the controlled release is a short termrelease. In one embodiment, the short term release is between 30minutes-1 hour. In one embodiment, the short term release is between 1hour-3 hours. In one embodiment, the short term release is between 3hours-10 hours. In one embodiment, the short term release is between 10hours-24 hours. In one embodiment, the controlled release is a long termrelease. In one embodiment, the long term release is between 24 hours-36hours. In one embodiment, the long term release is between 3 days-7days. In one embodiment, the long term release is between 7 days-1month. In one embodiment, the long term release is between 1 month-6months. In one embodiment, the long term release is between 6 months-1year. In one embodiment, the long term release is at least one year. Inone embodiment, the bone surgery comprises bone replacement including,but not limited to, hip replacement or knee replacement. In oneembodiment, the bone surgery comprises bone reconstruction. In oneembodiment, the bone surgery comprises bone fracture repair. In oneembodiment, the soluble signaling factor comprises MIM1. In oneembodiment, the surface bound signaling factor comprises EphrinB2 and/orTGF-β.

In one embodiment, the present invention contemplates a kit comprising acontainer comprising at least one artificial osteoclast cell suspendedin a pharmaceutically acceptable vehicle. In one embodiment, the kitfurther comprises instructions for using the artificial osteoclast cellto induce bone healing in a patient. In one embodiment, the artificialosteoclast cell comprises at least one encapsulated soluble osteoblastsignaling factor. In one embodiment, the soluble signaling factorcomprises MIM1.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a patient, wherein said patient comprisesat least one inflammatory symptom; ii) a microparticle comprising atleast one soluble Treg factor; and b) administering the microparticle tothe patient under conditions such that the at least one inflammatorysymptom is reduced. In one embodiment, the at least one soluble orsurface bound Treg factor comprises a regulatory T cell stimulatoryfactor. In one embodiment, the at least one soluble or surface boundTreg cell factor comprises a regulatory T cell inducing factor. In oneembodiment, the at least one soluble or surface bound Treg cell factorcomprises a regulatory T cell chemoattractant factor. In one embodiment,the patient has under gone periodontal surgery. In one embodiment, thepatient further comprises a tissue injury. In one embodiment, thepatient further comprises a periodontal disease. In one embodiment, thepatient comprises a cancer disease. In one embodiment, the microparticleis porous. In one embodiment, the microparticle is biodegradable,wherein the Treg chemoattractant factor undergoes controlled release. Inone embodiment, the controlled release is a short term release. In oneembodiment, the short term release is between 30 minutes-1 hour. In oneembodiment, the short term release is between 1 hour-3 hours. In oneembodiment, the short term release is between 3 hours-10 hours. In oneembodiment, the short term release is between 10 hours-24 hours. In oneembodiment, the controlled release is a long term release. In oneembodiment, the long term release is between 24 hours-36 hours. In oneembodiment, the long term release is between 3 days-7 days. In oneembodiment, the long term release is between 7 days-1 month. In oneembodiment, the long term release is between 1 month-6 months. In oneembodiment, the long term release is between 6 months-1 year. In oneembodiment, the long term release is at least one year. In oneembodiment, the Treg chemoattractant factor comprises CCL22 and/orvasoactive intestinal peptide.

DEFINITIONS

The term “artificial particle” or “artificial construct” as used herein,refers to any fabricated degradable polymer construct that is capable ofemulsification to encapsulate soluble factors, wherein the polymer maybe functionalized to attached surface bound factors. The constructs maythen manipulate native regulatory cells to induce immunologicaltolerance.

The term “artificial presenting cell” as used herein, refers to anyartificial particle or artificial construct that further comprises animmunological synapse.

The term “immunological synapse’ as used herein, refers to any specific,non-random, arrangement of biological compounds (i.e., for example,antibodies or soluble proteins) on a biological antigen presenting cellthat is complementary to a contacting region on a target cell (i.e., forexample, a lymphocyte). For example, an antigen presenting cell maycomprise an immunological synapse that resembles a “bull's eye” and hasa complementary contact region on a regulatory T cell (T_(reg) cell).

The term, “artificial dendritic cell” or “artificial APC” as usedherein, refers to any fabricated degradable, artificial presenting cellusing microparticle-based controlled release technology capable ofinteracting with a regulatory T cell. For example, cell-sized PLGAmicroparticles may be used to encapsulate soluble factors that arecapable of inducing regulatory T cells to induce immunosuppression. Forinvestigational studies, the soluble factors may be labeled withmonoclonal antibodies (i.e., for example, fluorescent labels).

The term “artificial osteoblast cell”, as used herein, refers to anyfabricated degradable microparticle having controlled release technologycapable of interacting with an osteoclast cell. For example, cell-sizedPLGA microparticles may be used to encapsulate soluble factors that arecapable of inducing osteoclasts to initiate bone healing. Forinvestigational studies, the soluble factors may be labeled withmonoclonal antibodies (i.e., for example, fluorescent labels).

The term “artificial osteoclast cell”, as used herein, refers to anyfabricated degradable microparticle having controlled release technologycapable of interacting with an osteoblast cell. For example, cell-sizedPLGA microparticles may be used to encapsulate soluble factors that arecapable of inducing osteoblasts to modulate bone healing. Forinvestigational studies, the soluble factors may be labeled withmonoclonal antibodies (i.e., for example, fluorescent labels).

The term “soluble regulatory T cell stimulating factors” as used herein,refers to any biological agent (i.e., for example, a protein, hormone,drug etc.) capable of interacting with T_(reg) cells that may becomeencapsulated within a polymer-based microparticle (i.e., for example, anartificial antigen presenting cell) and undergo controlled releaseduring the degradation of the microparticle. Such soluble factors maybe, for example, CCL22, IL2, or TGFβ.

The term “soluble osteoclast signaling factor” as used herein, refers toany biological agent (i.e., for example, a protein, hormone, drug etc)capable of interacting with osteoclast cells that may becomeencapsulated within a polymer-based microparticle (i.e., for example, anartificial osteoblast cell) an undergo controlled release during thedegradation of the microparticle. Such soluble factors may be forexample, MIM-1.

The term, “surface bound regulatory T cell stimulating factors” as usedherein, refers to any biological agent (i.e., for example, a protein,hormone, drug etc.) capable of interacting with T_(reg) cells, that areattached to the surface of a polymer-based microparticle (i.e., forexample, an artificial antigen presenting cell) and are not releasedduring the degradation of the microparticle. Such surface factors maybe, for example, CD3 antibody, CD28 antibody, or adhesion molecules.

The term, “surface bound osteoclast signaling factors” as used herein,refers to any biological agent (i.e., for example, a protein, hormone,drug etc.) capable of interacting with osteoclast cells that areattached to the surface of a polymer-based microparticle (i.e., forexample, an artificial antigen presenting cell) and are not releasedduring the degradation of the microparticle. Such surface factors maybe, for example, RANK-L.

The term “biomimetic” as used herein, refers to any artificial orsynthetic construct that has a similar biological effect as a nativebiological compound. Such similar effects may be due to similarities issize, shape, or specific (i.e., non-random) display of receptors,antibodies, or other active biological compounds. Such shapes include,but are not limited to, a circle, a square, a rectangle, a triangle, atrapezoid, a hexagon, an octagon, or a tetrahedron.

The term, “microparticle” as used herein, refers to any microscopiccarrier to which a compound or drug may be attached. Preferably,microparticles contemplated by this invention are capable offormulations having controlled release properties.

The term “PLGA” as used herein, refers to mixtures of polymers orcopolymers of lactic acid and glycolic acid. As used herein, lactidepolymers are chemically equivalent to lactic acid polymer and glycolidepolymers are chemically equivalent to glycolic acid polymers. In oneembodiment, PLGA contemplates an alternating mixture of lactide andglycolide polymers, and is referred to as a poly(lactide-co-glycolide)polymer.

The term “biocompatible”, as used herein, refers to any material doesnot elicit a substantial detrimental response in the host. There isalways concern, when a foreign object is introduced into a living body,that the object will induce an immune reaction, such as an inflammatoryresponse that will have negative effects on the host. In the context ofthis invention, biocompatibility is evaluated according to theapplication for which it was designed: for example; a bandage isregarded a biocompatible with the skin, whereas an implanted medicaldevice is regarded as biocompatible with the internal tissues of thebody. Preferably, biocompatible materials include, but are not limitedto, biodegradable and biostable materials.

The term “biodegradable” as used herein, refers to any material that canbe acted upon biochemically by living cells or organisms, or processesthereof, including water, and broken down into lower molecular weightproducts such that the molecular structure has been altered.

The term “controlled release” as used herein, refers to the escape ofany attached or encapsulated factor at a predetermined rate. Forexample, a controlled release of a factor may occur resulting from thepredicable biodegradation of a polymer particle (i.e., for example, anartificial antigen presenting cell). The rate of biodegradation may bepredetermined by altering the polymer composition and/or ratio'scomprising the particle. Consequently, the controlled release may beshort term or the controlled release may be long term. In oneembodiment, the short term release is between 30 minutes-1 hour. In oneembodiment, the short term release is between 1 hour-3 hours. In oneembodiment, the short term release is between 3 hours-10 hours. In oneembodiment, the short term release is between 10 hours-24 hours. In oneembodiment, the long term release is between 24 hours-36 hours. In oneembodiment, the long term release is between 3 days-7 days. In oneembodiment, the long term release is between 7 days-1 month. In oneembodiment, the long term release is between 1 month-6 months. In oneembodiment, the long term release is between 6 months-1 year. In oneembodiment, the long term release is at least one year.

The term “emulsification” as used herein, refers to any process thatmixes together at least two compounds that are not naturally miscibletogether. For example, an emulsification process may utilize agents(i.e., emulsifiers) that facilitate the co-solubility of immisciblecompounds. For example, emulsification may include methods that usetechniques including, but not limited to, precipitation, emulsions, meltcasting, spray drying, crystallization, shearing, or milling.

The term “polymer” as used herein, refers to any compound of highmolecular weight derived either by the addition of many smallermolecules, such as PLGA, or by the condensation of many smallermolecules with the elimination of water, alcohol, or the like.

The term “functionalized polymer” as used herein, refers to any polymerwere the terminal moiety has been chemically altered to facilitate theattachment of other chemicals and or compounds (i.e., for example,proteins, antibodies, hormones, drugs, etc.). For example, the terminalmoiety may include, but is not limited to, a carboxylic acid, an amine,a sulfide, a hydroxyl etc.

The term “immobilization” as used herein, refers a state where abiological compound is fixed in position as a complex and is unable tomove without the movement of the complex.

The term “attached” as used herein, refers to any interaction between amedium or carrier and a compound. Attachment may be reversible orirreversible. Such attachment may be, but is not limited to, covalentbonding, ionic bonding, Van de Waal forces or friction, and the like. Acompound is attached to a medium or carrier if it is impregnated,incorporated, coated, in suspension with, emulsified with, in solutionwith, mixed with, etc.

The term “covalent bond” as used herein, refers to a close associationbetween at least two atoms, wherein the atoms share electrons inspecific atomic orbital paths.

The term “non-covalent bond” as used herein, refers to a closeassociation between at least two atoms, wherein the atoms do not shareelectrons.

The term “patient” as used herein, is a human or animal and need not behospitalized. For example, out-patients, persons in nursing homes are“patients.”

The term “tissue transplant” as used herein, refers to any replacementof a tissue and/or organ within an individual with a similar tissueand/or organ from a different individual. In some cases, the individualsare from the same species. In other cases, the individual are fromdifferent species.

The term “immunological tolerance” as used herein, refers to anymodification of the immune system wherein specific antibodies may not beproduced, but the immune system remains responsive to other antigens.For example, specific immune related cells including, but not limitedto, T_(reg) cells, osteoclasts, and/or osteoblasts may be stimulated toinduce immunosuppression. Such “immunological tolerance” may also becapable of controlling autoimmune diseases including, but not limitedto, arthritis, Type I diabetes. Such “immunological tolerance” may alsobe capable of controlling inflammatory diseases including, but notlimited to, periodontal disease.

The term “administered” or “administering” a compound or drug, as usedherein, refers to any method of providing a compound or drug to apatient such that the compound or drug has its intended effect on thepatient. For example, one method of administering is by an indirectmechanism using a medical device such as, but not limited to a catheter,spray gun, syringe etc. A second exemplary method of administering is bya direct mechanism such as, oral ingestion, transdermal patch, topical,inhalation, suppository etc.

The term, “supramolecular activation clusters (SMACs)” as used herein,refer to a topologically complex, and discretely organized, arrangementof antigen presenting cell surface proteins and/or soluble proteins.SMACs may be comprised of a plurality of regions having localizeddensity of specific surface proteins and/or soluble proteins (i.e., forexample, central SMAC (cSMAC), peripheral SMAC (pSMAC), or distal SMAC(dSMAC). In general, the surface proteins comprise cell surfaceantibodies and/or receptors, while the soluble proteins comprise ligandsdirected to activating certain lymphocytes (i.e., for example, aregulatory T cell).

The term “apical region” as used herein, refers to the top of a sphereand spreading circumferentially from between approximately 1 degree to30 degrees from a polar axis. In one embodiment, the apical region mayspread circumferentially from between approximately 5 degrees to 20degrees from a polar axis. In one embodiment, the apical region mayspread circumferentially from between approximately 10 degrees to 15degrees from a polar axis.

The term “tropical region” as used herein, refers to a circumferentialregion of a sphere located between approximately 30-70 degrees from apolar axis. In one embodiment, the tropical region may spreadcircumferentially from between approximately 35 degrees to 60 degreesfrom a polar axis. In one embodiment, the tropical region may spreadcircumferentially from between approximately 40 degrees to 50 degreesfrom a polar axis.

The term “equatorial region” as used herein, refers to a circumferentialregion of a sphere located between approximately 70-90 degrees from apolar axis. In one embodiment, the equatorial region may spreadcircumferentially from between approximately 75 degrees to 85 degreesfrom a polar axis. In one embodiment, the equatorial region may spreadcircumferentially from between approximately 87 degrees to 93 degreesfrom a polar axis.

The term “random” as used herein, refers to a stochastic arrangement ofobjects formed without definite aim, purpose, method, or adherence to aprior arrangement; or in a haphazard way.

The term “non-random” as used herein, refers to a non-stochasticarrangement of objects with a definite aim, purpose, method and adheresto a prior conceived pattern.

The term “chemoattractant factor” as used herein, refers to any compoundand/or molecule that induces movement of chemotactic cells in thedirection of its highest concentration. For example, a chemoattractantfactor may include, but is not limited to, CCL22 and/or vasoactiveintestinal peptide (VIP).

The term “chemotactic cells” as used herein, refers to any biologicalcell exhibiting chemotaxis, wherein the chemotactic cells direct theirmovements according to certain chemicals in their environment.

BRIEF DESCRIPTION OF THE FIGURES

The patent application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing (s) will be provided by the office upon request andpayment of the necessary fee.

FIG. 1 provides exemplary data showing a T_(reg) proliferative doseresponse induced by degradable particles with randomly attached CD3antibodies and CD28 antibodies (ratio=1:1 or 1:4 cells/particle).Proliferation was measured by tritiated thymidine incorporation.Controls are equivalent amounts of soluble antibody (control). Inset:FITC labeled CD3 and CD28 antibodies for visualization),

FIG. 2 presents an illustrative embodiment showing how regulatory Tcells (T_(regs)) are attracted to: (A) a tumor; or (B) an artificialpresenting cell (i.e., for example, a synthetic dendritic cell) near thesurface of an allograft.

FIG. 3 shows a fluorescent image of a representative “bull's eye”immunological synapse orientation from a biological dendritic cell.Grakoui et al., Science 285:221 (1999).

FIG. 4A illustrates one embodiment of a method for fabricatingartificial presenting cell construct patterns that mimic a cellularimmunological synapse (IS). Step 1: Particles are immersed in PDMS orResin; Step 2: Etch so that only the top of the particles are exposedand chemically attach ligand (see arrows); Step 3: Etch remainder awayand label remainder of particle by chemically attaching 2^(nd) ligand(see arrows); and Step 4: Repeat-or-wash and collect.

FIG. 4B presents an exemplary fluorescent photomicrograph image of asynthetic dendritic cell comprising a central supramolecular activationcomplex (cSMAC) region (green/yellow) and a peripheral supramolecularactivation complex (pSMAC) region. See, Top Image arrows, respectively.Scanning Electron Microscopic backscatter imaging shows a bright, andintentionally over-labeled, cSMAC region (See, Bottom Image, whitearrow=gold-bead labeled protein).

FIG. 5A presents an illustration of one embodiment for the fabricationof polymer microparticles via a double-emulsion/solvent evaporationtechnique. Step 1: Sonication of a drug in an aqueous solution andpolymer in organic solution to form a primary emulsion; Step 2: Theprimary emulsion is poured into stirring detergent solution to form asecondary emulsion; and Step 3: The secondary emulsion is stirred andsolvent evaporates to form solid particles.

FIG. 5B presents exemplary data of the technique shown in FIG. 5A usinga freeze fracture scanning electron micrograph of microparticlesdemonstrating the interior, aqueous protein factor containingcompartments.

FIG. 6 illustrates standard carbodiimide chemistry to create an aminereactive site on the surface of PLGA microparticles starting with acarboxylic acid generated from the degradation of ester bonds.

FIG. 7 presents one embodiment to label functionalized PLGA polymersusing a novel compound (i.e., for example, 2-pyridyldithioethylamine) toconvert a carboxylic acid group into a thiol reactive group (See, middlepanel). A subsequent cleavage reaction liberates a detectable productupon conjugation which can be used to determine the extent of surfacelabeling (see, right panel).

FIG. 8 illustrates one embodiment of an in vitro chemotaxis assay.Microparticles containing chemokines are placed in the bottom of atranswell filter plate with T_(reg) cells isolated from a transgenicB6/GFP+ mouse on top of the opaque filter (left). After time, chemokineis released and T_(reg) cells migrate through the filter and up theconcentration gradient of chemokine (right). Fluorescence readings aretaken with a top and bottom detector plate reader to determine theextent of chemotaxis.

FIG. 9A presents exemplary data showing a release profile of CCL22 fromporous particles showing approximately linear release for over twenty(20) days.

FIG. 9B presents exemplary data showing the successful creation ofmicroparticles encapsulating CCL22 as used in FIG. 9A. The top portionshows a representative image of an intact microparticle, indicating theparticle's structural integrity and confirming the size. The bottomportion shows a representative image of a fractured particle confirmingthe porous nature of the particle created by the double emulsionencapsulation process.

FIG. 10 presents exemplary data showing CCL22 labeling (red) using themicroparticles in accordance with FIG. 9.

FIG. 11 presents an illustration of one embodiment comparing healthilygum tissue (left side) with gum tissue affected by periodontal disease(right side).

FIGS. 12A and 12B present exemplary data showing the effect ofartificial antigen presenting cells encapsulating CCL-22 on periodontaldisease in a mouse model. AA: infected, untreated mice. LP: infected,aAPC-CCL22 treated mice; CP: infected, aAPC-only mice.

FIG. 13A presents an illustration representing a hypothesized couplinginteraction for osteoclast stimulation. For example, osteoclasts may berecruited by MCSF and activated by surface-bound RANK-L and/or OSCAR-L.

FIG. 13B presents an illustration of an artificial osteoblast cell thatmimics the coupling interactions shown in FIG. 13A.

FIG. 14A presents an illustration representing a hypothesized couplinginteraction for osteoblast stimulation. For example, osteoblasts arerecruited by MIM1 and activated by surface-bound TGF-beta and EphrinB2(BMP2).

FIG. 14B presents an illustration of an artificial osteoclast cell thatmimics the coupling interactions shown in FIG. 14A.

FIG. 15 presents representative scanning micrographs of CCL22-containingmicroparticles.

FIG. 15A shows the external porous structure of a microparticle.

FIG. 15B shows the internal structure of the microparticle displayingthe inner pockets created by a water-in-oil emulsion.

FIG. 16 presents exemplary data showing release kinetics of CCL22 frommicroparticles as measured in physiological buffered saline (PBS). ErrorBars=Standard Deviations; n=3.

FIG. 17 presents exemplary data showing a representative volume of anaveraged sized distribution of microparticles comprising CCL22determined by average volume diameter measurements. Error Bars=StandardError of the Mean; n=6.

FIG. 18 presents exemplary data showing CCR4 expression and in vitrotranswell migration assay.

FIG. 18A: Histograms depicting the absence of CCR4 expression on naïveCD4+ T cells but its presence on activated CD4+ T cells, as determinedby flow cytometry.

FIG. 18B: Transwell migration assays show significantly greatermigration of activated regulatory and effector CD4+ T cells whencompared to respective populations of naïve cells at differentconcentrations of CCL22. (*p<0.05, one-tailed Students ‘t’ test)

FIG. 18C: Expression of CCL22 is enhanced in CD4+ cells in the presenceFoxP3 in comparison to the absence of FoxP3.

FIG. 19 presents exemplary data showing an in vivo migration of Tregstowards microparticles comprising CCL22.

FIG. 19A shows a representative schematic describing one experimentaldesign for live animal imaging. Luc+ Treg indicates a Treg populationisolated from transgenic mice constitutively expressing the luciferasegene.

FIG. 19B presents exemplary data showing representative fluorescence andluminescence images obtained at day 7 post injection, fluorescenceimages were used to outline areas of particle localization (i.e., forexample, with Pro Living Image® 2.60.1) and these outlines weresuper-imposed onto luminescence images taken with the mouse in the exactsame position to demonstrate co-localization of Tregs and CCL22microparticles.

FIG. 19C presents exemplary data showing kinetics of Treg migrationtowards the injection sites; average radiance measurements obtained fromthe luminescence images are displayed as a ratio of CCL2 microparticlesto Blank microparticles (BlankMP). Error Bars=Standard Deviation; n=3.

FIG. 20 presents exemplary data of non-invasive live animal imaging. Theleft limb was injected with BlankMP-680 and the right limb was injectedwith CCL22MP-680. Representative fluorescence (left) and luminescence(right) images taken from a single mouse at different time pointsfollowing injection. Days 4 & 7: Significant co-localization of Tregwith CCL22 microparticles. Days 2 and 9: Very little or noco-localization of Treg with CCL22 microparticles. Values represent thetotal flux (photons/sec) of fluorescence or luminescence intensities.ROI (regions of interest) were drawn automatically using Igor Pro LivingImage® 2.60.1. Influx of Treg only towards CCL22MP was observed overmultiple experimental cycles (n=6). Over time it was observed that Treglocalized in mucosal tissues such as the gut and the lung, which secreteCCL22 endogenously. Endogenous Treg migration to the hind limbs wasnegligible and the cells that migrated to these sites were specificallytowards CCL22MP.

FIG. 21 presents one method of preparing microparticles.

FIG. 22 presents exemplary data showing mRNA expression of markers inperiodontal tissues resected from diseased mice (A) FoxP3 a marker forTregs. (B) Antiinflammatory, pro-regenerative cytokine IL-10. (C) Boneresorbing cell (ostecoclast) activation factor RANKL. (D) EndogenousCCL-22 (Treg recruiting) chemokine.

FIG. 23 presents exemplary data showing abrogation of alveolar boneresorption in a mouse model of periodontitis. (A-B) dissectingmicroscope images of resected maxilla mechanically de-fleshed and soakedin dispase overnight. Dotted-red boxes outline large differences inalveolar bone levels (between the internal control B LEFT, and CCL-22 MPtreated B RIGHT). (A) Representative image from mice that receivedBlankMPs A RIGHT in the right maxilla and no treatment (internalcontrol) A LEFT in the left maxilla. (B) Representative image from amouse that received CCL-22 MPs B RIGHT in the right maxilla and notreatment (internal control) B LEFT in the left maxilla (C, right) Areameasurements (CEJ-ABC) for the right maxilla of mice from each treatmentand control group. *p-value<0.05

FIG. 24 presents exemplary data showing tumor regression after inductionof Treg cells. Transgenic luciferase expressing lewis lung carcinomacells (derived from C57Bl/6 mice) were injected into FVB mice 4 dayspost particle injections. Quantitative live animal imaging was performedsubsequently at regular time intervals. Above images show qualitativedata obtained at 2 different time points, suggesting that the CCL22microparticles are able to slow down rejection of cellularallo-transplants.

FIG. 25 presents tumor regression kinetic data from the experimentdemonstrated in FIG. 24. * Indicates p<0.05; Wilcoxon rank-sum test fornull hypothesis that normalized luminescence for CCL22MP is same as thatof BlankMP or bolus CCL22. # p≦0.05 (comparison between CCL22MP andBlankMP only). n=11 BlankMP and CCL22MP (n=5 for bolus CCL22)

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to the field of inducing immunologicaltolerance by specifically manipulating immune-related cells. Suchimmunological tolerance may be induced by providing biomimeticartificial particles that bypass native immunological cells. Forexample, biomimetic artificial particle compositions are contemplatedwhich comprise soluble factors that activate specific immune-relatedblood cells, including, but not limited to, macrophages and/ormonocytes. Such factors may comprise chemoattractant factors. Thesebiomimetic artificial particles may further present a specificbiomimetic surface pattern that results in a targeted cell response suchthat the particles represent artificial presenting cells.

The present invention contemplates artificial particles such that asynthetic construct may be capable of presenting the samereleased/secreted and/or chemically-bound/plasma membrane-boundcompounds (i.e., for example, soluble proteins, antibodies, receptors,hormones etc.) as actual living cells, and is not limited to modulatingthe immune system (i.e., for example, modulation of bone healing, tissueinflammation and/or tumor regression). The present inventioncontemplates various embodiments wherein these artificial constructsreplace biological cell-cell interactions within the processes of manybiological functions including, but not limited to, wound healing,restoration from a diseased state, or enhancement in normal function ofcellular processes.

In one embodiment, the present invention contemplates an investigationalplatform capable of testing the in vivo roles and efficiencies of bothsoluble secreted and surface bound T_(reg) cell factors. Such Treg cellfactors may including but not limited to stimulatory factor, inducingfactor, or chemoattractant factors. In one embodiment, the platformcomprises an in vivo testing platform (i.e., for example, an in vivomouse model). In one embodiment, the platform comprises an in vitrotesting platform (i.e., for example, a primary cell culture). Thesemodels are capable of evaluating various combinations of membranesurface factors and/or soluble releasable factors that are attached toand/or encapsulated within, a biodegradable, microparticle constructthat stimulates T_(reg) cells and induces allograft tolerance. Althoughit is not necessary to understand the mechanism of an invention, it isbelieved that these biodegradable microparticle constructs may act as“artificial antigen presenting cells” that comprise a biomimeticimmunological synapse and encapsulate and controllably release solublefactors that stimulate T_(reg) cell stimulation, proliferation, andchemotaxis. Hoffmann et al., Blood 104, 895-903 (2004); Green et al.,Proc Natl Acad Sci USA 100: 10878-10883 (2003); Curiel et al., Nat Med10:942-949 (2004); and Belghith et al., Nat Med 9:1202-1208 (2003).Further, the constructs are capable of presenting surface-boundmonoclonal antibodies (mAb) specific for T_(reg) cell surface activationfactors.

I. Immunological Tolerance

In developing a strategy to replace and/or decrease post-transplantimmunosuppressant therapy, “reprogramming” the immune system toselectively accept a foreign antigen without an systemic immunedown-regulation is seen as a desirable option. This state may bereferred to as a permanent immunological tolerance. One modulator ofimmunological tolerance is suspected to be regulatory T-cells (T_(reg)).T_(reg) cells have been shown to be capable of antigen-independentimmune suppression and can be activated by immature or tumor-manipulateddendritic cells, thereby inducing immunosuppression. In vitro, T_(reg)cells respond to several known soluble factors and even latex beadscoated with monoclonal antibodies specific for several T_(reg) cellsurface receptors. Tumors actively secrete compounds to modulateregulatory T-cells so as to evade immune recognition. Although notintuitively obvious, it is believed that the induction of permanentallograft tolerance may be achieved by mimicking tumor-inducedimmunosuppression.

CD4+CD25+ regulatory T-cells are believed to express the CCR4 receptorwhich binds to the chemokine CCL22. CCL22 was reported to be involved inthe migration of regulatory T-cells to tumor sites because tumors alsoactively secrete CCL22. Curiel et al., Nat Med 10:942-949 (2004). Thisrepresents just one of the methods that tumors have for evading immunerecognition. Evasion of an immune response provides for tumor survivalgiven that many of its basic biological features such as geneticinstability, invasive growth, and tissue disruption are inherentlypro-inflammatory. Besides attracting regulatory T-cells to its vicinity,tumors are believed to also have the ability to influence biologicalantigen presenting cells (i.e., for example, biological dendritic cells)to down-regulate processing and presentation of tumor associatedantigens, inhibit co-stimulatory expression (i.e. retain immaturephenotype), and alter their cytokine secretion profile towards immunetolerance. Pardoll, D., Annual Review of Immunology 21:807-839 (2003).It has been reported that tumors also secrete TGF-β, which may influenceregulatory T-cell mediated tolerance. Chen et al., Proc Natl Acad SciUSA 102:419-424 (2005). Although it is not necessary to understand themechanism of an invention, it is believed that these other mechanismssimilar to those of tumor immune evasion and survival may also be usefulin inducing allograft tolerance.

Control over the induction of immunological tolerance would affordpermanent allograft acceptance, bypassing a lifelong regimen ofimmunosuppressive agents. Although it is not necessary to understand themechanism of an invention, it is believed that this tolerance can bemediated by activating suppressive regulatory lymphocytes (i.e., forexample, regulatory T cells).

In one embodiment, the present invention contemplates atherapeutically-relevant, modular platform to deliver multiple solublefactors and membrane surface-bound factors. In one embodiment, thesefactors are delivered in vivo by artificial particles into the vicinityof regulatory T cells (T_(reg) cells). In one embodiment, the deliveredfactors modulate T_(reg) cell proliferation. In one embodiment, thedelivered factors modulate T_(reg) cell immunosuppressive capacity. Inone embodiment, the delivered factors induce Treg cell chemotaxis. Inone embodiment, the platform is delivered in vivo. In one embodiment,the platform comprises a biocompatible and/or biodegradable syntheticconstruct, such as an artificial antigen presenting cell (aAPC). In oneembodiment, the aAPC is approximately the size of a biological cell. Inone embodiment, the aAPC comprises covalently-bound monoclonalantibodies and/or encapsulated soluble cytokines/chemokines. In oneembodiment, the antibodies and cytokine/chemokines stimulate lymphocyteproliferation. In one embodiment, the lymphocyte stimulation isperformed in vitro. In one embodiment, the lymphocyte stimulation isperformed in vivo.

In one embodiment, the present invention contemplates a constructcomprising a pattern of membrane surface factors such that the patternmimics a biological dendritic cell/regulatory T-cell immunologicalsynapse (IS). In one embodiment, the pattern increases the stimulatorycapacity of the construct. In one embodiment, the synapse comprises a“bull's-eye” pattern. In one embodiment, the “bull's-eye” patterncomprises an apical region and an equatorial region. In one embodiment,the pattern further comprises a region between the apical and equatorialregions.

In one embodiment, the present invention contemplates a method forcreating an artificial biomimetic immunological synapse. In oneembodiment, the synapse is discrete and topologically complex. In oneembodiment, the synapse comprises at least two immunosuppressivefactors. In one embodiment, the synapse is located on the surface of anartificial presenting cell. In one embodiment, the synapse comprises a“bull's-eye” pattern. In one embodiment, the “bull's-eye” patterncomprises an apical region and an equatorial region. In one embodiment,the pattern further comprises a region between the apical and equatorialregions.

A. Tolerogenic Cells

The biological processes whereby tolerance is induced involve thesuppression or deletion of alloreactive T-cells which would otherwisedestroy an allograft. This process is thought to be mediated by a subsetof regulatory T-cells which represent 5-10% of all peripheral CD4+lymphocytes. Mahnke et al., Blood 101:4862-4869 (2003).

Suppressive lymphocyte involvement in immune response was firstsuggested in the early 1970s. Gershon et al., Immunology 21:903-914(1971). Recently, however, a suppressive regulatory T-cell (T_(reg)) hasbeen implicated in the development of immune tolerance. For example, apopulation of these lymphocytes that express high levels of CD25, the αsubunit of the IL2 receptor, have been reported as an immune suppressor.Sakaguchi et al., J Immunol 155:1151-1164 (1995).

T_(reg) cells also express a unique transcription factor called FoxP3,which is thought to be required for the development of T_(reg)suppressive capacity and is a recognized marker for T_(reg) cells. Horiet al., Science 299:1057-1061 (2003); and Fontenot et al., Nat Immunol4:330-336 (2003). Furthermore, it is believed that the extent ofexpression of this transcription factor can correlate with thesuppressive capacity and activation state of a T_(reg) cell. It has beenshown that transfer of the gene for FoxP3 into cells which are CD4+CD25−confers regulatory capacity which is otherwise absent. Fontenot et al.,Nat Immunol 4:330-336 (2003). T_(reg) cells may also express high levelsof CTLA-4 (i.e., for example, CD152) which may also be involved in theirregulatory capacity as this molecule can bind to the B7 class ofco-stimulatory molecules in place of CD28 thereby resulting in theproduction of transforming growth factor-β, or TGF-β. Perez et al.,Immunity 6:411-417 (1997). TGF-β, along with engagement of the T-cellreceptor, has been demonstrated to differentiate naïve, peripheralCD4+CD25− T-cells into CD4+CD25+ cells with suppressive capacity,suggesting that this factor may be important for the in vivo generationand maintenance of T_(reg) cells. Walker et al., J Clin Invest112:1437-1443 (2003). Regulatory T-cells are also believed to expresschemokine receptors including, but not limited to, CCR4 and CCR8,rendering them fully capable of migration (i.e., for example, bychemotaxis) to a site of inflammation or to the lymph nodes uponappropriate signaling. Iellem et al., J Exp Med 194:847-853 (2001).

Mechanisms of action used by regulatory T-cells in promoting tolerancein vivo has been a topic of dispute. Some believe that cell-to-cellcontact between T_(reg) cells and the alloreactive lymphocytes providethe activation stimulus that results in immunosuppression. Othersbelieve that specific stimulatory factors (i.e., for example, solublefactors and/or membrane surface bound factors) which have been shown tobe secreted from regulatory T-cells may be responsible forcontact-independent suppression (i.e., for example, TGF-β, IL-10,CTLA-4, and IL-4). For example, studies which support the latterhypothesis demonstrate that: i) T_(reg) activation by TNF-β may beblocked by monoclonal antibodies (Belghith et al., Nat Med 9:1202-1208(2003); and ii) T_(reg) activation is diminished in TGF-β receptorknock-out mice (Green et al., Proc Natl Acad Sci USA 100:10878-10883(2003)). However, it has been reported that suppression by T_(reg) cellsin vitro is not inhibited by blocking TGF-β and suggest a cellularcontact dependant mechanism. Piccirillo et al., J Exp Med 196:237-246(2002).

Nevertheless, one interesting notion regarding the method of T_(reg)cell suppression which is gaining popular acceptance is an antigenindependent suppression mechanism, known as “bystander regulation”.Several new reports are providing evidence for “bystander regulation”which opposes a previous hypothesis that regulatory T-cellspreferentially mediate immunosuppression in an antigen specific manner.Graca et al., Proc Natl Acad Sci USA 101:10122-10126 (2004); and Karimet al., Blood 105:4871-4877 (2005). In particular, Karim et al. reportedthat CD25+CD4+ T_(reg) cells generated by nominal antigens under coverof anti-CD4 monoclonal antibody treatments were fully capable oftolerizing mice to cardiac allografts comprised of completely differentantigens. Therefore, although most studies show that T-cell receptorstimulation seems to be involved in tolerization, antigen specificitymay or may not be limiting in the overall effects of immunosuppressioninduced by regulatory T-cells. The data presented herein suggest thatT_(reg) cell immunosuppression may be meditated through a combination ofcontact dependant and independent mechanisms.

1. Antigen Presenting Cells

In one embodiment, the present invention contemplates methods that wouldreconcile the apparently conflicting in vitro and in vivo data regardingcytokine-dependent suppressive capacity. In one embodiment, the methodcomprises using cell types that are not present in the in vitroexperimental models (i.e., for example, a co-mediator cell). Although itis not necessary to understand the mechanism of an invention, it isbelieved that these co-mediator cells may secrete their ownimmunomodulating cytokines or act in other suppressive capacities at thesame time as regulatory T-cells. For example, a co-mediator cell maycomprise an antigen presenting cells (APCs). Antigen presenting cellsare also referred to as dendritic cells and have been shown to have atolerogenic capacity. Matzinger et al., Nature 338:74-76 (1989);Steinman et al., Proc Natl Acad Sci USA 99:351-358 (2002); Brocker etal., J Exp Med 185:541-550 (1997); and Volkmann et al., J Immunol158:693-706 (1997).

APCs and lymphocytes may interact at a contact point in accordance witha “two signal” model of stimulation. For example, the presence of T-cellreceptor (TCR)/Major Histocompatibility Complex (MHC) engagement (i.e.,for example, Signal 1) without the presence of a proper co-stimulatorymolecule engagement (i.e., for example, Signal 2) may lead to lymphocyteprogression toward a state of anergy or deletion through apoptosis. Insupport of this hypothesis, it has been demonstrated that engagement ofthe T-cell receptor using monoclonal antibodies specific for CD3 (i.e.,for example, T-cell receptor subunit believed responsible for signaltransduction), in the absence of co-stimulation, led to long lastingproliferative unresponsiveness in murine T-cell clones. Jenkins et al.,J Immunol 144: 16-22 (1990).

Dendritic cells (i.e., for example, APCs) in an immature state (i.e.that have not been stimulated by inflammatory factors) have beenobserved to possess a low level expression of co-stimulatory moleculeswhile still maintaining expression of MHC Class I and II. These immaturedendritic cells have a high capacity to take up material byreceptor-mediated endocytosis, pinocytosis, and phagocytosis and arethought to survey the periphery and constantly process and presentself-antigen. These observations suggest that an immunological tolerantstate is maintained in the absence of co-stimulatory molecules even whenself-antigen is persistently presented. Consequently, it would bereasonable to believe that the presence of co-stimulatory moleculesrepresent danger signals. Matzinger, P. Science 296:301-305 (2002).Although it is not necessary to understand the mechanism of aninvention, it is believed that this would explain why autoimmunitydevelops when the normal, perpetual tolerant state is somehow disrupted.

In light of the above, it is most likely not a coincidence thatpersistent expression of antigen in the periphery is also thought to besufficient for generation of regulatory T-cells. Taams et al., Eur JImmunol 32:1621-1630 (2002). Furthermore, soluble factors are secretedby T_(reg) cells that generate and maintain APCs in an immature stateincluding, but not limited to: i) IL10 (De Smedt et al., Eur J Immunol27:1229-1235 (1997)); ii) TGF-β(Geissmann et al., J Immunol162:4567-4575 (1999)); and IL4 (Inaba et al., Journal of ExperimentalMedicine 176:1693-1702 (1992)). On the other hand, others have shownthat immature/tolerogenic dendritic cells also have the ability tomediate contact-dependent stimulation of T_(reg) cells. These secretedsoluble factors are also suggested to generate and maintain T_(reg)cells thereby creating the possibility for feedback regulation. Steinmanet al., Annu Rev Immunol 21:685-711 (2003). This feedback mechanismmight even extend to the apoptosis of dendritic cells which are alreadyactivated or mature by regulatory T-cells. Frasca et al., J Immunol168:1060-1068 (2002). These data suggest that APCs and T_(reg) cellsinteract to modulate immunotolerance.

The current understanding of biological processes whereby regulatoryT-cells (T_(reg)) promote tolerance is presented in Table 1.

TABLE 1 Summary Of T_(reg) Observations Observation SignificanceReference Tolerogenic T_(reg) cells express These markers can aid in theSakaguchi et al., J Immunol high surface levels of CD4 & isolation ofT_(reg) cells 155: 1151 (1995). CD25. The transcription factor Foxp3Activation state of T_(reg) can be Fontenot et al., Nat Immunol ishighly expressed in determined by measuring 4: 330 (2003). activatedT_(reg). Foxp3. TGF-β1 can generate T_(reg) TGF-β1 is important toBelghith et al., Nat Med while inhibiting other T-cells. preferentialdevelopment of T_(reg). 9: 1202 (2003); and Green et al., PNAS 100:10878 (2003). T_(reg) express the chemokine Chemokines such as CCL22Chen et al., American receptors CCR4 and CCR8 may be used topreferentially Journal of Transplantation and are attracted by theirrecruit T_(reg). 6: 1518 (2006); and ligands. Curiel et al., Nat Med 10:942 (2004). Tolerogenic dendritic cells Contact-mediated and solubleSteinman et al., Annu Rev have been associated with, and factorstimulation provided by Immunol 21: 685 (2003). have the capacity tostimulate, these APCs can generate T_(reg) T_(reg). T_(reg) stimulatedby a particular T_(reg) are capable of promoting Karim et al., Bloodantigen can mediate immune allograft tolerance regardless 105: 4871(2005). suppression in the presence of of the antigen/TCR-stimulus anallograft without this used to generate them. antigen. Beads coated withmAb A similar strategy could be Hoffmann et al., Blood specific to CD3and CD28 in utilized in vivo by including 104: 895 (2004). the presenceof IL2 were soluble factor(s) in a capable of stimulating andbiodegradable format. proliferating T_(reg) in vitro.

Although it is not necessary to understand the mechanism of aninvention, it is believed that the stimulation, and behavior of, T_(reg)cells can generate immune tolerance. Professional antigen presentingcells (APCs) (i.e., for example, dendritic cells: DCs) have also beenshown to have tolerogenic capacity, although the precise mechanism oftheir suppressive function is unclear. Matzinger et al., Nature 338:74(1989); and Steinman et al., Proc Natl Acad Sci USA, 99:351 (2002). Arelationship between DCs and T_(reg) cells has been observed where apersistent presentation of antigen by tolerogenic DCs generates T_(reg)cells through both contact-mediated and secreted soluble signal-mediatedstimulation. Steinman et al., Annu Rev Immunol 21:685 (2003). Thus, APCsmight mediate immunosuppression by stimulating T_(reg) cells through adual pronged mechanism.

Although it is not necessary to understand the mechanism of aninvention, it is believed that an artificial particles comprising Tregcell factors should modulate T_(reg) cells in a manner similar tobiological antigen presenting cells. For example, a membrane surfacepresentation (i.e., for example, an immunological synapse) ofstimulating protein factors on a biological dendritic cell is notdisplayed in a random pattern. In one embodiment, the present inventioncontemplates an artificial antigen presenting cell comprising solubleand/or membrane bound stimulating factors displayed in non-randompatterns.

In particular, biological antigen presenting cells have been reported todevelop a mature immunological synapse (IS) and are composed ofdiscretely defined spatial regions of receptors. Monks et al., Nature395:82 (1998). Some in the art have termed these regions assupramolecular activation clusters, or SMACs. Grakoui et al., Science285:221 (1999). A central SMAC (cSMAC) region may comprise areasproviding factors for TCR stimulation and/or soluble factors forco-stimulation. The cSMAC may be surrounded by at least one peripheralSMAC (pSMAC) region that are usually rich in adhesion molecules (i.e.,for example, ICAM). Further, pSMACs may be surrounded by a distal SMAC(dSMAC) region that may have, for example, a high CD45 density. Using adual probe fluorescence imaging technique, the SMAC spatial organizationresembles a “bulls-eye”. See, FIG. 3.

2. In vitro T_(reg) Stimulation

In vitro testing with primary, regulatory T-cells provides a way todetermine if the soluble factors encapsulated within an artificialparticle are biologically active. Preferred assays are directly relatedto the biological activity of the encapsulated factors with respect totheir modulatory effects on T_(reg) cells. In vitro primary T_(reg)cells also provide a testing platform for the optimization of artificialparticles in preparation for therapeutic in vivo administration.

A method for large scale in vitro expansion of T_(reg) cells has beenreported. Hoffmann et al., Blood 104:895 (2004). In this study, theproliferation and suppressive capacity of cultured T_(reg) cells wasincreased using latex beads randomly coated with soluble IL2 andCD3/CD289 monoclonal antibodies. It was unreported if the T_(reg) cellpopulations produced by Hoffmann et al. are functionally the same asthose produced in vivo by biological antigen presenting cells.Sufficient discrepancies are observed between in vitro and in vivosuppressive mechanisms to indicate that they are not functionally thesame. Belghith et al., Nat Med 9:1202 (2003); and Green et al., PNAS100:10878 (2003). Such a problem in the art identifies a need to studyin vivo T_(reg) cell activation. A significant disadvantage of theHoffmann et al. technique, is that the random distribution stimulatoryfactors on the latex beads did not present the T_(reg) cells with anSMAC IS complex.

Nonetheless, in vitro data is provided herein demonstrating thestimulatory capacity of synthetic dendritic cells by several subsets ofT-cells. For example, primary lymphocytes were harvested and incubatedwith synthetic dendritic cells and the resulting level of activationwere measured.

Commercially available cell isolation kits (i.e., for example, MACS) maybe used to harvest CD4+CD25+ T_(reg) and CD4+CD25− helper T-cells fromlymph nodes of B6 mice. Once incubated with artificial antigenpresenting cells, regulatory T-cells can be examined for proliferativecapacity using techniques including, but not limited to, tritiatedthymidine incorporation, CSFE dilution, or Flow Cytometry. Cytokinesecretion profiles (i.e., for example, IL-2 or IFN-gamma) may bedetermined using ELISPOT. To assess regulatory T-cell activation thepresence of the intracellular transcription factor, FoxP3, may also bemeasured in permeabilized cells using Flow Cytometry.

To verify the exclusivity of T_(reg) cell stimulation, artificialantigen presenting cells can also be incubated with other lymphocytepopulations (CD4+CD25−, and CD8+) which are then tested for knownactivation markers. Although it is not necessary to understand themechanism of an invention, it is believed that because of the release ofTGF-β1 by aAPCs these non-suppressor cells will not be stimulated andmay actually even gain suppressive capacity. Fu et al., Am J Transplant4:1614 (2004). It is further believed that the removal of CD28co-stimulation may result in anergy of helper T-cells. Ragazzo et al.,PNAS 98:241 (2001). Thus, it is not presumed that a cSMAC region mustcontain both membrane bound TCR stimulation factors and solubleco-stimulation factors simultaneously to achieve preferential regulatoryT-cell activation.

Nonetheless, T_(reg) cells may be studied in vitro using artificial APCscomprising multiple membrane surface-bound stimulatory factors andsoluble secretable stimulatory factors. For example, the activity ofencapsulated soluble stimulatory factors may be tested using primaryT_(reg) cells in vitro by assays measuring effects on celldifferentiation, cell proliferation, and/or chemotaxis. In someembodiments, a plurality of aAPC formulations may be used wherein eachaAPC formulation comprises a different amount of both surface boundfactors and soluble releasable factors. In one embodiment, the presentinvention contemplates a method comprising identifying an optimizedformulation.

3. In Vivo Effects of Artificial Particles on Regulatory T Cells(T_(regs))

In one embodiment, the present invention contemplates a methodcomprising implanting artificial particles in vivo wherein T_(reg) cellsare recruited and/or activated. In one embodiment, the T_(reg) cellrecruitment and/or activation induces biological graft tolerance.Although it is not necessary to understand the mechanism of aninvention, it is believed that the biological graft tolerance is aresult of T_(reg) cell induced immunosuppression.

Before T_(reg) cells can be directly manipulated by an artificialparticle, however, they need to be attracted to an artificial particle.In one embodiment, the present invention contemplates an artificialparticle comprising an encapsulated chemotactic factor (i.e., forexample, CCL22). Alternatively, the T_(reg) cell population may beincreased by performing a T_(reg) cell adoptive transfer. This procedureis especially useful during investigational and/or techniqueverification studies wherein the transferred T_(reg) cells are labeledwith green fluorescent protein (i.e., for example, collected fromtransgenic GFP+ B6 mice). The resulting enriched T_(reg) population invivo facilitates histological and flow cytometry analyses. Further, anoptimization of the chemotactic factor is most likely necessary becauseof inherent differences between in vivo and in vitro techniques, asdiscussed above.

The embodiments of the present invention contemplate manytransplantation models for use as testing models. In some embodiment,testing models include, but are not limited to, skin allograft orheterotopic heart transplants, and are used to compare high and lowamounts of donor derived antigen presenting cells. These donorbiological particles may interfere or enhance the function of theartificial particles. However, it should be theoretically feasible todrastically outnumber the biological antigen presenting cells throughadministration of multiple depot injections near the transplantationsite and/or coating the allograft with artificial particles. In oneembodiment, a skin allograft testing model is chosen due to thesimplicity of the procedure and the ability to use a control syngenicgraft on the same animal. In one embodiment, a heterotopic hearttransplant testing model is chosen, not only due to its relativelysimple procedure and method of data collection, but also because of theconsistency in the rejection profile as opposed to liver and kidneytransplants.

In one embodiment, the present invention contemplates a methodcomprising measuring regulatory T cell migration and activationfollowing exposure to synthetic, biodegradable constructs. In oneembodiment, the method comprises an in vivo murine model. This modelprovides unexpected and unpredictable advantages over traditional invitro models, because, as discussed elsewhere, the behavior of T_(reg)cells in vitro is not believed to be equivalent to that observed invivo. M. Belghith et al., Nat Med 9:1202 (September, 2003); Green etal., PNAS 100:10878 (2003).

In one embodiment, the present invention contemplates a testing methodcomprising co-implanting a tissue transplant graft and a plurality ofartificial particles in vivo, wherein the artificial particles recruitand activate the T_(reg) cells to induce a permanent graft tolerance.For example, artificial particles may be tested for the ability torecruit and stimulate regulatory T-cells by injecting artificialparticles into mice which have been adoptively transferred with GFP+T_(reg) cells. Imaging studies can then easily identify in vivolocalizations of the GFP+ T_(reg) cell following artificial particleinjection. Further, various artificial particle formulations may beco-administered at the time of tissue transplantation (i.e., forexample, skin allograft or heterotopic heart transplantation) to examinetheir therapeutic ability to induce allograft tolerance.

In one embodiment, the present invention contemplates an in vivo methodcomprising manipulating T_(reg) cells using synthetic dendritic cells.In one embodiment, the synthetic dendritic cells stimulate T_(reg),thereby resulting in immunosuppression. Although it is not necessary tounderstand the mechanism of an invention, it is believed that thatsynthetic dendritic cells which are approximately 10 μm do not move fromthe site of implantation given that they are too large to cross theendothelial barrier or redistribute via the reticuloendothelial system.In one embodiment, the method further comprises administering syntheticdendritic cells with encapsulated soluble factors and/or surface factorsinto a first leg flank of a B6 mouse. In one embodiment, theadministering comprises injection. In one embodiment, the injectioncomprises a subcutaneous injection. In one embodiment, the injectioncomprises an intramuscular injection. In one embodiment, a second legflank is injected with a synthetic dendritic cell without encapsulatedsoluble and/or surface factors (i.e., for example, as a negativecontrol). At 1 Day or 3 Days after the injection(s), mice will beeuthanized and skin or muscle will be resected for histological analysis(i.e., for example, staining for T_(reg) cells and Foxp3). Wang et al.,Am J Transplant 6:1297 (June, 2006). The sensitivity of this protocolcan be enhanced by adoptively transferring fluorescent T_(reg) isolatedfrom GFP+ B6 mice into normal B6 mice to increase the number ofdetectable cells available for chemotaxis.

In one embodiment, the present invention contemplates a methodcomprising prolonging the survival of a skin allograft using abiomimetic immunosuppressive synthetic dendritic cell.

4. T_(reg) Cells And Transplant Tolerance

The generation of T_(reg) suppressor cells has been attempted in vivoprior to transplantation with the goal of inhibiting allograftrejection. Most of these methods involve the administration of analloantigen, with the final result being an increase in the number andactivation state of regulatory T-cells. In mice, it has been shown thatnaïve regulatory T-cells still have suppressive capacity, but 10 foldgreater quantities are required to promote graft acceptance whencompared to populations that have been stimulated prior totransplantation with alloantigen. Graca et al., J Immunol 168:5558-5565(2002). Furthermore, administration of this alloantigen is usuallyaccompanied by CD4 monoclonal antibodies to avoid eliciting a hostileimmune response. Kingsley et al., J Immunol 168:1080-1086 (2002).

Other monoclonal antibodies which block co-stimulation pathways may alsobe used to generate regulatory T-cells including, but not limited to,monoclonal antibodies that block CD154-CD40 engagement. van Maurik, A.,Herber, M., Wood, K. J. & Jones, N. D. (2002) J Immunol 169, 5401-4;Taylor et al., Blood 99:4601-4609 (2002). Also, CD-3 monoclonalantibodies were used to generate regulatory T-cells via stimulationthrough the T-cell receptor (TCR) and was reported to control theprogression of diabetes. Herold et al., N Engl J Med 346:1692-1698(2002). Native, immature dendritic cells may be capable of generatingCD4+CD25+ T_(reg) cells in vivo if alloantigen is specifically targetedto them by an antibody which binds to DEC205; a receptor involved in therecycling of antigen through late endosomal compartments. Mahnke et al.,Blood 101:4862-4869 (2003).

Ex vivo expansion and manipulation of tolerogenic cells holds muchpromise for therapeutic application given their relatively low frequencyin peripheral tissues. For this potential to be realized, this expansionwould have to maintain an appropriate regulatory phenotype, includingthe ability to migrate to the correct sites of inflammation and thelymphoid compartments. Attempts at generating regulatory T-cells invitro have involved stimulation with alloantigen with co-stimulants,such as: i) vitamin D3 and dexamethasone (Barrat et al., J Exp Med195:603-616 (2002)); ii) IL10 (Levings et al., J Exp Med 193:1295-302(2001)); iii) TGF-β (Yamagiwa et al., J Immunol 166:7282-7289 (2001));and iv) IL2 (Taylor et al. Blood 99:3493-3499 (2002)). Transfection ofT-cell subsets with the gene for FoxP3 in vitro has also endowedregulatory capacity. Fontenot et al., Nat Immunol 4:330-336 (2003).

II. Biomimetic Artificial Antigen Presenting Cells

In one embodiment, the present invention contemplates a methodcomprising fabricating cell-sized, degradable particles (i.e., forexample, a synthetic dendritic cell or artificial antigen presentingcell). In one embodiment, the particles release soluble factors. In oneembodiment, the particles display surface bound factors in a non-randompattern. In one embodiment, the particles display surface bound factorsin a random pattern. In one embodiment, the non-random pattern simulatesan immunological synapse. In one embodiment, the synapse comprises asupramolecular activation complex.

In one embodiment, the artificial particle comprises a biodegradableconstruct thereby providing a controlled release of incorporated and/orattached compounds (i.e., for example, therapeutic agents, antibodies,cytokines, or chemokines). In one embodiment, the particle comprises adegradable polyester including, but not limited to,poly(lactic-co-glycolic) acid (PLGA). PLGA has been used in FDA-approvedgrafts, sutures, and/or drug delivery microparticulates such as LupronDepot®. Degradable PLGA polymer microparticles are superior toconventional latex or polystyrene “artificial APCs” because PLGA confersbiodegradability. Further, unlike latex and polystyrene polymerparticles that only allow surface attachment of proteins, PLGA polymerparticles. allow encapsulation of protein cell factors (i.e., forexample, IL2, TGF-β, and CCL22) through a double emulsion/solventevaporation procedure. Odonnell et al., Advanced Drug Delivery Reviews28:25-42 (1997). Further, a controlled release of soluble cell factorsfrom PLGA polymers can be engineered to create an appropriate localconcentration of these cell factors, which would be accompanied bycell-to-cell contact with immobilized molecules on the particle surface.Such immobilized molecules (i.e., for example, a monoclonal antibody)can be easily bound to the particle through a streptavidin-biotinlinkage using several established chemical techniques. Further, thecontrolled release of encapsulated one such soluble protein factor fortwenty days from microparticles has been demonstrated (i.e., forexample, CCL22). See, FIG. 9A. Scanning electron microscopy confirmedthe porous nature of the microparticles responsible for the controlledrelease characteristics. See, FIG. 9B.

Artificial stimulation using randomly coated micron-sized sphericalconstructs to promote cytotoxic T-cell receptor engagement along withco-stimulation has been used to gain insight into the role of IL2 as asoluble stimulation factor. Mescher et al., J Immunol 149:2402-2405(1992); and Curtsinger et al., J Immunol Methods 209:47-57 (1997). Whenapplied to regulatory T-cells, stimulation by randomly coated constructsprovided substantial increases in total cell count, but also enhancedsuppressive capabilities over non-stimulated naïve CD4+CD25+ cells.Others have shown that randomly coated particles produce expanded andactivated regulatory T-cells were polyclonal, and maintained expressionof appropriate chemokine receptors. Hoffmann et al., Blood 104:895-903(2004).

Nevertheless, these references using randomly coated particles do notcontemplate the specific advantages of creating and using non-randomlypatterned artificial presenting cells that specifically mimic biologicalcell surface protein patterns (i.e., for example, a supramolecularactivation complex). In one embodiment, the present inventioncontemplates fabricating an artificial tolerogenic dendritic celldesigned to stimulate a regulatory T-cell. In one embodiment, these cellsized constructs present protein factors capable of both cooperating inTCR engagement and releasing T_(reg) cell co-stimulatory moieties. Forexample, the constructs are able to contain and controllably releasesoluble chemokines and proliferation promoting factors. These constructshighly flexible, being capable of modification for both the type, andlevel, of surface bound and/or soluble releasable stimulating factors.In some embodiments, the use of biodegradable materials will provideuses of these “artificial dendritic cells” for in vivo diagnostics toidentify the role of individual T_(reg) cell stimulating factors, andtherapeutically to promote allograft tolerance.

A. The Particle Core

In one embodiment, the present invention contemplates a particlecomprising CD3 antibodies and CD28 antibodies non-randomly attached tothe surface of a poly-lactic-co-glycolic acid (PLGA) particle. In oneembodiment, the antibodies are attached by absorption. In oneembodiment, the antibodies are attached covalently. In one embodiment,the particles are covalently surface modified with streptavidin. In oneembodiment, the particles comprise biotinylated mAb, wherein the mAbsstimulate T_(reg) cells. In one embodiment, the attached antibodies arein a biologically active state. See, FIG. 1.

These biodegradable particles have numerous advantages over T_(reg) cellmodulators known in the art, for example: a) particle biodegradabilityand biocompatibility allow for in vivo investigational and therapeuticuse for controlling T_(reg) cells in immune suppression; or 2) suchparticles are capable of controllably releasing soluble proteins such ascytokines and chemokines from the particle interior while alsopresenting immobilized protein factors on their surface; and 3)surface-mediated stimulation and release of soluble factors from thesame artificial construct closely mimics T_(reg) cell stimulation bybiological dendritic cells.

In one embodiment, the particles comprise releaseable soluble factorsthat preferentially activate different T-cell subsets. In oneembodiment, the releasable factors are encapsulated within theparticles. Although it is not necessary to understand the mechanism ofan invention, it is believed that the releasable factors arecontrollably released subsequent to particle degradation. In oneembodiment, the factor comprises a suppressive cytokine (i.e., forexample, TGF-β12). In one embodiment, the factor comprises aT_(reg)-preferential cytokine (i.e., for example, CCL-226). Theseparticles behave similarly to tumor tissues that are believed toactively suppress immune responses by selectively secreting TGF-β12and/or CCL-226 to attract T_(reg)s to their vicinity. Otherwise, abody's normal immune response might otherwise destroy the malignancy.Curiel et al., Nat Med 10:942 (2004); and Chen et al., Proc Natl AcadSci USA 102:419 (2005). Although it is not necessary to understand themechanism of an invention, it is believed that the particlescontemplated by the present invention are capable of both delivering theimmobilize surface stimulation factors and the secreted solublestimulation factors to act in a similar manner as tolerogenic biologicaldendritic cells (i.e., for example, APCs) to influence T_(reg) mediatedimmunoresponsivity. See, FIG. 2.

B. Artificial Antigen Presenting Cell Surface Protein Patterns

Recent studies have attempted to study IS orientations in vitro usingmicrofabricated, patterned “dots” of CD3 antibody (i.e., providing TCRstimulation) in a field of ICAM adhesion molecules. Doh et al., ProcNatl Acad Sci USA 103:5700 (2006). In vitro models have distinctdisadvantages, however, because T_(reg) cell stimulation is morepronounced when using a spherical structure as opposed to a flatsurface, such as a cell culture dish. Curtsinger et al., J ImmunolMethods 209: 47 (1997). Further, the in vitro approach provided inCurtsinger et al. does not allow simulation for the study of all three(3) SMAC regions in a mature IS. Especially lacking in Curtsinger et al.is the ability to study biomimetic SMAC regions. Although recent datademonstrate patterns having protein orientations using two (2) factorson a particle surface, a method of precisely orienting a pattern of >2factors on a particle has yet to be discovered. Chen et al., Proc NatlAcad Sci USA 104:11173 (2007).

In one embodiment, the present invention contemplates a methodcomprising creating a synthetic dendritic cell surface comprising abiomimetic IS pattern. See, FIG. 4. In one embodiment, the IS patterncomprises a cSMAC region. In one embodiment, the IS pattern comprises apSMAC region. In one embodiment, the IS pattern comprises a dSMACregion. Although it is not necessary to understand the mechanism of aninvention, it is believed that this method is analogous to drainingwater out of a tub filled with bowling balls. After only a fraction ofthe liquid has drained, the top portion of the ball (having a circularshape, if viewed from above) will have been exposed (thereby making thesurface available for a first modification) while the remaining portionis still covered by the water (not available for a first modification).After further draining, an annular ring comprising the firstmodification and the remaining portion is exposed, thereby making theremaining portion available for labeling with a second factor, therebycreating a second annular ring. This process may be repeated as manytimes as desired limited only by the size of the ball and the width ofthe multiple annular rings.

A method for patterning a prototype particle with multiple factor layerswas demonstrated using poly-dimethylsiloxane (PDMS). As is known fromphotolithography techniques, PDMS can be etched by the application of asolvent. This is analogous to partial draining of water from a bathtubfull of bowling balls. Using PDMS, a prototype synthetic dendritic cellcomprising a “bull's eye” IS was made by surface etching an artificialcSMAC region surrounded by artificial pSMAC region. See. FIG. 4. In oneembodiment, the present invention contemplates a method for stimulatingT_(reg) cells using “bull's eye” patterned IS-mimetic particles, whereinthe stimulation is qualitatively and quantitatively superior torandomly-labeled synthetic dendritic cells.

The above construction of prototype synthetic dendritic cells using duallabels definitively shows preferential labeling in different particleregions. Nonetheless, some factor overlap is present indicated byoverlapping fluorescent signals (i.e., a yellow central regionindicating the presence of both labels) and some gold-bead labeling inthe SEM images outside of the intended central region. Theseobservations may be a result of uneven PDMS etching which may beimproved by using techniques that are more easily controllable withrespects to speed, distance, and residual matter. For example, a sodiumsilicate (“liquid-glass”) system can provide a more easily controlledetching process by using<5% hydrofluoric acid (HF) evenly applied at acontrollable rate of approximately 100 nm per minute resolution (datanot shown).

C. Functional Protein Patterns on Synthetic Dendritic Cell Prototypes

The present invention contemplates compositions comprising a fabricatedcell-sized, biodegradable particle exhibiting secretable soluble factorsand immobilized surface factors displayed in a biomimetic pattern.

In one embodiment, the present invention contemplates a syntheticdendritic cell comprising PLGA microparticles. In one embodiment, thepresent invention contemplates a method of producing a syntheticdendritic cell comprising encapsulating soluble and/or surface factorsinto the PLGA microparticles using one of many emulsification and/orencapsulation procedures (i.e., for example, double emulsion). In oneembodiment, the synthetic dendritic cells comprise microparticlediameters of approximately 1-1000 μm. In one embodiment, themicroparticle diameters are approximately 5-500 μm. In one embodiment,the synthetic dendritic cells comprise microparticle diameters ofapproximately 10-50 μm. Lu et al., J Biomed Mater Res 50:440 (2000).Encapsulation methods are commonly used to load agents into polymermatrices that are capable of releasing factors by modifying parametersincluding, but not limited to, copolymer ratios and/or molecular weight.Odonnell et al., Advanced Drug Delivery Reviews 28:25 (1997).

Encapsulation loading of synthetic dendritic cells can be easilyverified by dissolution techniques using sodium hydroxide and/ordimethylsulfoxide followed by protein detection assays such asbicinchoninic acid (BCA) or enzyme linked immunosorbent assay (ELISA) todetect total protein. Fluorescent surface factor labeling determineswhich construction method (PDMS vs. “liquid glass”) produces the mostconsistent results. For example, three separate labels can be appliedusing a bioconjugate strategy, for example: i) a first exposed regionmay be protected with small-molecule biotinylation; ii) a second exposedregion may be protected with an asymmetric disulfide (for subsequentthiol exchange) 20; and iii) a third exposed region may be protectedwith standard N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC) chemistry.

In one embodiment, the present invention contemplates a method forevaluating three (3) exposed particle regions by fabricating theparticles using different formulations. In one embodiment, a formulationcomprises cSMAC (CD3 mAb)+pSMAC (ICAM) (2 labels/2 regions) forcomparison to formulations created on a flat surface (i.e., for example,an in vitro cell culture dish). In one embodiment, a formulationcomprises cSMAC (CD3+CD28 mAbs)+pSMAC (ICAM) (3 labels/2 regions) forcomparison with randomly displayed CD3 mAbs and CD28 mAbs particles. Inone embodiment, a formulation comprises cSMAC (CD3/CD28)+pSMAC(ICAM)+dSMAC (CD45 mAb) for comparison with a complete syntheticimmunological synapse comprising at least three (3) SMACs.

III. Controlled Release of Compounds from Artificial Particles

Controlled release of compounds from microparticles (i.e., for example,PLGA microparticles) fabricated from the double emulsion procedureprovide many advantages as an artificial construct for releasingencapsulated compounds as opposited to more traditional compounds suchas latex or styrene based particles. PLGA microparticle advantagesinclude, but are not limited to: i) the double emulsion procedure allowsfor encapsulation of any number of water soluble activation factors; ii)particle size can be easily adjusted based on known fabricationparameters; iii) PLGA is non-toxic, biodegradable, and has propertieswhich allow it to be tuned to adjust release of the encapsulated agents;and 4) the surface of PLGA particles can be easily labeled using EDC(carbodiimide) chemistry.

In one embodiment, the present invention contemplates a methodcomprising encapsulating and controllably releasing soluble factorswhile maintaining relatively stable surface presentation of monoclonalantibody. Although it is not necessary to understand the mechanism of aninvention, it is believed that T_(reg) cells are attracted towards anartificial particle, engage surface protein stimulation factors, andreceive the proper proliferation and maturation signals from secretedsoluble proteins at the same time. In one embodiment, the encapsulatingcomprises at least one soluble secretable factors, including but notlimited to, IL2, TGF-β, and CCL22. These factors may be encapsulatedindividually or in any combination. In one embodiment, the releasing iscontrolled by using different molecular weight PLGA or through otherfabrication parameters including, but not limited to, drug distribution,occlusion radius, amorphicity/crytallinity of the polymer, excipientsetc. Rothstein et al., J Materials Chem 18:1873-1880 (2008). Anempirical process determines the final amounts of factors to beencapsulated given that the appropriate quantity of these factors foroptimal stimulation of regulatory T-cells in vivo is yet still unknown.

In one embodiment, the particle surface is covalently labeled todetermine the number of functionalized sites capable of attaching theimmobilized protein factors. In one embodiment, the labeling comprises2-pyridyldithioethylamine. In covalent labeling of the particle, itwould be desirable to allow for positioning of the surface mediatedregulatory T-cell factor(s) (i.e., for example, monoclonal antibodiesfor CD3 or CD28) in a way which does not covalently link the activesite. To achieve this, some embodiments conjugate streptavidin to theparticle surface. Although it is not necessary to understand themechanism of an invention, it is believed that at least one ofstreptavidin's four active binding sites for biotin will be exposed.Consequently, an artificial particle may be labeled using commerciallyavailable biotinylated antibodies specific for many commerciallyavailable surface molecules. Alternatively, streptavidin may bethiolated (i.e., for example, by using readily available products suchas NHS esters of S-acetylthioacetic acid or Trauts reagent) andintegrated into the 2-pyridyldithioethylamine labeling embodimentdescribed herein.

In some embodiments, biodegradable polymers pre-labeled with biotin maybe used in fabrication of artificial particles. For example, poly(lacticacid) (PLA) copolymers with poly(ethylene glycol) (PEG) are reported tobe conjugated with biotin. Cannizzaro et al., Biotechnol Bioeng58:529-535 (1998). Microparticles have been made from these polymershave been reported to produce microparticles having surface bindingsites using emulsion/solvent-evaporation techniques. Further, themicroparticle surface was successfully labeled using an excess ofneutravidin followed by biotinylated monoclonal antibodies and proteins.Sakhalkar et al., Proc Natl Acad Sci USA 100:15895-15900 (2003).

IV. Release Characteristics of CCL22

The data presented herein demonstrates that a sustained release of CCL22was achieved by loading the chemokine into degradablepoly(lactic-co-glyoclic) acid (PLGA)—based microparticles (CCL22MP)using a water-oil-water double emulsion-evaporation technique. Zhao etal., Biomaterials 26:5048-5063 (2005). Scanning electron micrographs(SEM) of intact microparticles indicates that these microparticles arespherical and slightly porous. FIG. 15A. SEM particle cross-sectionsalso show a plurality of internal structures formed during creation in awater-in-oil primary emulsion. FIG. 15B. The surface of a microparticlecomprising CCL22 (CCL22MP) was specifically formulated to be porous, toallow continuous release of chemokine (i.e., for example, releaseoccurring without intermittent periods of lag). Rothstein et al., J.Mater. Chem. 18:1873-1880 (2008); Rothstein et al., Biomaterials30:1657-1664 (2009). For example, release experiments performed inphosphate-buffered saline demonstrate a constant release of CCL22 forover 3 weeks. See, FIG. 16. Further, the microparticles were designed tobe large enough (i.e., for example, >10 μm) to avoid their uptake byphagocytic cells and to prohibit their movement through capillaries,with consequent immobilization at the site of injection. FIG. 17.

Simultaneously, the ability of CCL22 to attract murine T cells wasexamined. Expression of chemokine receptors for CCL22 (i.e., forexample, the CCR4 receptor) are known to control the ability of cells torespond to CCL22 gradients. Moreover, there is evidence that onlyactivated T cells express the CCR4 receptor. Baatar et al., Journal ofImmunology 178: 4891-4900 (2007); Lim et al., Journal of Immunology177:840-851 (2006); and Lee et al., Journal of Experimental Medicine201:1037-1044 (2005); and FIG. 18A. The data presented herein show thatactivated T cells (both regulatory and effector cell populations), butnot naïve T cells, migrated towards a CCL22 gradient in vitro using atranswell chamber-based assay, a response that is consistent with theirsurface receptor expression patterns. FIG. 18B.

Having confirmed that activated Tregs migrate toward a CCL22 gradientand that controlled release formulations could be used to sustain therelease of CCL22, the capability of CCL22MPs to attract Tregs in vivowas tested. For example, CCL22-releasing polymeric particles wereinjected into the triceps surae of FVB mice followed by i.v. infusion ofex vivo activated, alloantigen-specific Tregs (AATregs) thatconstitutively expressed the luciferase gene. Following an activationstimulus (i.e., for example, an allogeneic-dendritic cell injection) themigration pattern of AATregs could be monitored by non-invasive liveanimal imaging. FIG. 19A. The data demonstrate that immediately afterallogeneic-dendritic cell stimulation (i.e., for example, approximately4-7 days after injection) a significantly greater number of AATregs wererecruited to the site of the CCL22MP injection when compared to aninternal control of microparticles lacking CCL22 (BlankMP). FIG. 19B andFIG. 20. Further, upon analyzing the kinetics of migration, it wasdetermined that cellular localization was transient and that within 7days of the stimulation event, no significant numbers of AATregpersisted at the site of particle injections. See, FIG. 19C.

One explanation of the data is that the decline in AATreg numbers may bedue to the absence of stimulatory/inflammatory signals at the site ofCCL22MP injection at that time. Presumably, the presence of Treg can beextended in a stimulatory microenvironment, in combination with othersurvival factors. The potential therapeutic implications of a degradablecontrolled release formulation capable of recruiting Tregs are manifold.For example, CCL22MP may be used in combination with an infusion ofcells expanded ex-vivo. Current pre-clinical data suggest that infusionof freshly-isolated or ex vivo-expanded Tregs can be used to preventrejection of organ transplants and to suppress autoimmune diseases, butchallenges such as obtaining adequate numbers and highly purifiedpopulations of Tregs have hindered progress into clinical trials. Bruskoet al., Immunological Reviews 223:371-390 (2008); and Riley et al.,Immunity 30:656-665 (2009). Given that the present data suggest thatTregs can be attracted to a local site using CCL22, it may be possibleto lower the numbers of injected cells, or potentially, to usepopulations with lower purity. In one embodiment, the present inventioncontemplates a method comprising a microparticle populationencapsulating CCL22, wherein a controlled release of CCL22 from themicroparticle improves tissue transplantation success (i.e., forexample, murine pancreatic islet cell transplantation). In oneembodiment, the released CCL22 attracts Tregs to the pancreatic isletcells. Zhang et al., Immunity 30:458-469 (2009).

Another use of CCL22-containing polymeric microparticles would be toattract naturally occurring Treg populations. If the numbers ofrecruited Tregs prove sufficient to control adverse immune responses atthe site of particle injection, then therapeutic effects could berealized without infusion of ex vivo-expanded Tregs. This hypothesis hasbeen reported within the context of a murine model of periodontitis, adisease that is associated with dissipation of Tregs from the gingivaltissues and loss of immune homeostasis. Garlet et al., Microbes andInfection 7:738-747 (2005). The data presented herein indicates thatadministration of CCL22MP leads to local recruitment of FoxP3+ cells tothe gingival tissues and corresponding reversal of adverse outcomesassociated with periodontitis (infra). Conventional methods ofCCL22-based sustained release have disadvantages in that this particularchemokine is known to attract both activated Tregs and activatedeffector T cells. FIG. 18; and Bromley et al., Nature Immunology9:970-980 (2008); Thus, in some cases it is quite possible that thenumbers of recruited Tregs may be insufficient to control the immuneresponse.

However, CCL22-based release formulations can easily be modified tosimultaneously release immunosuppressive agents that can suppress thefunctions of activated effector T cells in situ, thereby assisting theTregs in controlling adverse immune responses. Additionally, althoughCCL22 can attract both activated Tregs and effector T cells in vitro,studies in vivo suggest that CCL22 production and release associatedwith tumors or long-surviving allografts results in localimmunosuppression. Curiel et al., Nature Medicine 10:942-949 (2004); andLee et al., Journal of Experimental Medicine 201:1037-1044 (2005),respectively. Although it is not necessary to understand the mechanismof an invention, it is believed that this CCL22-associated effect may bedue to an enriched population of recruitable Tregs in the periphery whencompared to activated effector Tcells, or where equivalent numbers ofboth populations are recruited and the suppressive effect of Tregs isdominant.

In conclusion, the local attraction of Tregs in vivo usingchemokine-loaded sustained delivery vehicles has been demonstrated.Controlled release microparticle formulations as contemplated herein areparticularly useful as a modular platform for therapeutic development aswell as a tool to study Treg-dependent modulation of immune responses insitu.

V. CCL22 Microparticle Therapy

To verify that Tregs are recruited toward microparticles that sustainrelease of CCL-22, formulations were administered into the periodontiumof diseased mice. Briefly, mice were orally exposed to the periodontalpathogen A. actinomycetemcomitans (Aa) on day 0, and also receivedCCL-22 microparticles (CCL-22 MPs) or blank (empty) microparticles(BlankMPs) in their periodontal pockets. Specifically, mice receivedmicroparticle injections at day −1, 10 and 20, mice treated with VIPinjections received systemic injections at days −1, 7, 14, 21, 28.Untreated mice served as negative controls. At day 30, all mice weresacrificed and maxilla's were resected. Real-time polymerase chainreaction (QPCR) analysis provided strong evidence that our formulationsrecruit Tregs to a greater extent than other groups as detected bygreater expression of FoxP3. FIG. 22A. This same treatment group alsoshowed statistically greater expression of the pro-regenerative cytokineIL-10. FIG. 17B. Further, a decreased expression of bone resorbing cellactivator RANKL was observed. FIG. 22C. Interestingly, systemic VIPinjections induced endogenous production of CCL-22 presumably leading toTreg recruitment, and the disease symptom attenuation as demonstratedbelow.

To confirm that Treg recruiting formulations attenuate periodontaldisease symptoms, levels of alveolar bone resorption was examined inexperimental mouse periodontitis. G. P. Garlet et al., Clin Exp Immunol147, 128 (January, 2007); D. T. Graves, D. Fine, Y. T. Teng, T. E. VanDyke, G. Hajishengallis, J Clin Periodontol 35, 89 (February, 2008); andJ. J. Yu et al., Blood 109, 3794 (2007). To this end, B6 mice (n=6) wereinfected orally with the periodontal pathogen A. actinomycetemcomitans(Aa) and treated with microparticles containing CCL-22 (CCL-22 MPs) orempty microparticles (BlankMPs) according to the schedule outlinedabove. Additionally vasoactive intestinal peptide (VIP) was administeredintraperitoneally. Mice receiving only PBS and the thickenercarboxymethyl cellulose served as controls (Untreated). Experimentaltreatments were administered in the periodontal pocket of the rightmaxilla while left maxillae were used as internal negative controls ineach mouse. Alveolar bone loss was quantified as the area between thecementoenamel junction (CEJ) and the alveolar bone crest (ABC) FIG. 23,CEJ and FIG. 23A, ABC). Alveolar bone levels are shown on the left(internal control, FIG. 23A LEFT) and right (BlankMP treated, FIG. 23ARIGHT) maxilla. Both the internal control and BlankMP treated maxilla(FIG. 23A, both) exhibited significant alveolar bone loss. However, FIG.23 RIGHT depicts a maxilla treated with CCL-22 MPs, revealingsignificantly reduced alveolar bone loss, both compared to the internalcontrol maxilla (FIG. 28 LEFT) and the BlankMP treated maxilla (FIG. 23ARIGHT). Quantification of total bone loss (area in mm2) reveals thatCCL-22 MP as well as VIP (systemic IP injections) treatments led tosignificantly less alveolar bone resorption than BlankMPs. FIG. 23C.

VI. Clinical Applications

A. Immunosuppressant Therapy Substitution for Tissue/Graft Transplants

The development of new immunosuppressive agents has been the generaldirection in efforts to improve the transplantation success rate. Forexample, the use of agents which inhibit the production of lymphocyteproliferation factors have extended the half life of pediatric hearttransplants. Pietra et al., Prog Pediatr Cardiol 11:115-129 (2000).Presently, research is underway in efforts to further increase pediatricheart half-lives using agents which inhibit the de novo synthesis ofnucleotides and block co-stimulation of T-cells.

Presently available immunosuppressant drugs act to abrogate acuterejection of tissue transplants. Tissue transplant rejections areusually characterized by recognition of the foreign donor antigen by theadaptive immune system and the subsequent expansion of lymphocytes whichattack the transplanted tissue. Symptoms which accompany acute rejection(including, but not limited to, pain at the transplantation site orfever-like symptoms) are more frequent and severe immediately followingtransplantation, but commonly recur 6-12 months subsequent to theprocedure. Persistent episodes of acute rejection are thought to lead toa state of chronic rejection coupled with a failure of manyimmunosuppressant drugs. The most viable option following the onset ofchronic tissue rejection is re-transplantation, which is undesirablefrom both the standpoint of allograft availability and the increaseddifficulty of the repeated operation.

A major disadvantage of immunosuppressant therapy is that in order toavoid both episodes of acute rejection and the initiation of chronicrejection, immunosuppressant drugs must be administered over the entirelife of the transplant recipient. A further disadvantage ofimmunosuppressant therapy is the frequent incidence of side effects. Forexample, in the case of cyclosporine treatment (a commonly used agentwhich decreases the production of interleukin 2, or IL2), patients havebeen known to experience tremors, headaches, blurry vision, high bloodpressure, and inhibited kidney function. Other commonly usedimmunosuppressants also cause frequent side effects, which range fromgrowth retardation to behavioral instability to neurotoxicity. Besidesthese drug specific side effects, the consequences of long termimmunosuppression in general can be profound including, but not limitedto, increases in the risk of infection, heart disease, diabetes, andcancer.

While a combined administration of the immunosuppressant mycophenolatemofetil along with Vitamin D3 has been reported to generate dendriticcells with a tolerogenic phenotype resulting in increased numbers ofregulatory T-cells in mice, mycophenolate mofetil suffers from the sameadverse side effects as the immunosuppressants mentioned above and hasalso been implicated in causing birth defects in animals. Gregori etal., J Immunol 167:1945-1953 (2001). Furthermore, immunosuppressants cancause inhibition of regulatory T-cells as well as the T-cells thesetherapies intend to impede. Li et al., Nat Med 5:1298-1302 (1999). Dueto the potential of these tolerogenic cells, it is important that agreater understanding of the factors involved in the expansion andemployment of T_(reg) cells in vivo is developed.

The present invention contemplates alternatives to extended immunesystem down-regulation provided by immunosuppressant therapy. Suchalternatives are expected to provide a dramatic improvement over thestate-of-the-art in the clinic. For example, the most desirable of thesealternatives would render the patient's immune system in a state ofcomplete, permanent tolerance to a specific foreign body (i.e., forexample, a single antigen and/or antigen complex). Once this completeand permanent tolerance is attained, no further treatment is necessaryto prevent transplant-related adverse reactions. Furthermore, therecipient's immune system would otherwise function normally, being fullycapable of fighting pathogens and/or stopping the progression of tumorcells. Although it is not necessary to understand the mechanism of aninvention, it is believed that infants up to fourteen (14) months oldinnately have tissue tolerance due to the lack of soluble and/or surfacerejection factors which mediate immediate rejection of the transplant.As the infant's immune system matures, however, this innate tissuetolerance is lost. Fan et al., Nat Med 10, 1227-1233 (2004).

B. Implant Tolerance

Numerous polymeric biomaterials and metal materials are implanted eachyear in human bodies. Among them, drug delivery devices provide localtherapeutic effect for diseases which lack efficient treatments.Controlled release systems are in direct and sustained contact with thetissues, and some of them degrade in situ. Thus, both the materialitself and its degradation products must be devoid of toxicity. Theknowledge and understanding of the criteria and mechanisms determiningthe biocompatibility of biomaterials are therefore of great importance.

The classical tissue response to a foreign material leads to theencapsulation of the implant, which may impair the drug diffusion in thesurrounding tissue and/or cause implant failure. This tissue responsedepends on different factors, especially on the implantation site.Indeed, several organs possess a particular immunological status, whichmay reduce the inflammatory and immune reactions. Among them, thecentral nervous system is of particular interest, since many pathologiesstill need curative treatments.

In one embodiment, the present invention contemplates a method ofinducing implant tolerance by the administration of artificial antigenpresenting cells as described herein.

C. Tissue Healing

In one embodiment, the present invention contemplates using artificialmicroparticles comprising soluble and surface bound factors to inducetissue healing states including, but not limited to bone healing, woundhealing, and disease-induced injury tissue restoration.

1. Bone Healing

Over the past several decades, those having ordinary skill in the arthave been attempting to understand the interactions between the cellsthat mediate the process of bone remodeling and healing. It is believedthat the cells that mediate bone formation (i.e., for example,osteoblasts) may be capable of stimulating osteoclast cells. Osteoclastcells have been reported to resorb and remodel bone. Mundy G. R., Bone24(5 Suppl):35S-38S (1999). Osteoclasts, derived from a hemopoieticmonocyte cell lineage (e.g. in the same cell lineage as dendritic cellsand macrophages), resorb bone using proton and enzymatic secretion.Teitelbaum S. L., Science 289; 1504-1508. It is generally believed thatapproximately 10-15% of human bone is perpetually undergoing remodelingin order for our mineralized tissue to grow and adapt to stress.However, the current evidence suggests that when theosteoblast-osteoclast communication pathway breaks down, osteoscleroticsymptoms may occur including, but not limited to, fractures, severeinfections, blindness, deafness, deformities, or stroke.

Signaling factors (i.e., for example, soluble signaling factors andsurface-bound signaling factors) have been consistently implicated asintermediaries between osteoblasts and osteoclasts. Mundy G. R., Bone24(5 Suppl):35S-38S (1999). See, FIG. 13A. In one embodiment, thepresent invention contemplates a composition comprising anantibody-ligated OSCAR, wherein the antibody/OSCAR composition serves asa co-stimulus with the signaling factors. Merck et al., Journal ofImmunology 176(5):3149-3156 (2006). Although it is not necessary tounderstand the mechanism of an invention, it is believed that theantibody/OSCAR composition may be necessary but not sufficient forosteoclast activation alongside other surface-bound signals (i.e., forexample, RANK-L) and soluble signaling factor MCSF27.

Conversely, it is commonly known that secretion of osteoprotegrin (OPN)(a molecule that inhibits RANK-L signaling) can diminish osteoclasticactivity. Udagawa et al., Endocrinology 141:3478-3484 (2000); and Itohet al., Endocrinology 142:3656-3662 (2001). As dendritic cells mayundergo suppression via apoptosis/anergy (supra), a similar mechanismdownregulating the osteoblast-osteoclast interaction could beapplication to diseases where the degree of osteoclast resorptionexceeds that of osteoblast healing including, but not limited to,osteoporosis or osteopenia.

Finally, has been reported that a balance between bone resorption andhealing may be mediated not only by communication of osteoblasts toosteoclasts, but also by communication of osteoclasts to osteoblasts.Martin et al., Trends in Molecular Medicine 11: 76-81 (2005). Thiscross-talk is commonly referred to as “osteo-coupling”. Although boneresorption processes are believed to release soluble factors embedded inbone matrix that can recruit and differentiate osteoblastic precursors,some studies suggest that osteoclasts still strongly stimulateosteoblasts in the absence of resorption. Although it is not necessaryto understand the mechanism of an invention, it is believed that severalsoluble signaling factors and several surface-bound signaling factorsare likely to be involved.

In one embodiment, the present invention contemplates an artificialbiodegradable osteoblast cell that directly and indirectly interactswith an osteoclast cell. In one embodiment, the artificial osteoblastcell mimics osteo-coupling signaling that results in the modulation ofbone healing. In one embodiment, the artificial osteoblast cell acts asa biomimetic compound by providing surface bound RANK-L signalingfactors, anti-OSCAR antibodies, and a soluble signaling factor (i.e.,for example, MIM-1 and/or MCSF-27). Although it is not necessary tounderstand the mechanism of an invention, it is believed that theartificial osteoblast cell is capable of stimulating osteoclast cells toa greater degree than the “unorganized” forms because of a patternedorganization of the signaling factors. In other embodiments, asurface-bound signaling factor comprises osteoclastic differentiationfactor (ODF). ODF has been reported to act as a co-stimulator withRANK-L. Suda et al., Endocrinology Rev. 13:66-80 (1992). In otherembodiments, OPN is used to suppress osteoclasts. Udagawa et al.,Endocrinology 141:3478-3484 (2000).

In some embodiments, osteo coupling may involve a variety of signalingfactors. In one embodiment, a soluble signaling factor comprises MIM1, achemokine specific to osteoblast precursors. Falany et al., Biochemicaland Biophysical Research Communications 281(1):180-1805 (2001). In oneembodiment, a surface-bound signaling factor comprises EphrinB2, thatappears to stimulate osteogeneic differentiation. Zhao, et al., CellMetabolism 4:111-121 (2006). In one embodiment, a surface-boundsignaling factor comprises TGF-β, believed to stimulate osteoblasts.Pfeilschifter et al., Proceedings of the National Academy of Sciences84:2024-2028 (1987). Although it is not necessary to understand themechanism of an invention, it is believed that cell-sized biodegradableartificial osteoblast cells displaying surface bound signaling factors(i.e., for example, RANK-L, ODF, EphrinB2 and/or TGF-β) on a polymersurface and controllably releasing a soluble signaling factor (i.e., forexample, MIM1 and/or MSCF-27) might act, in combination, as “a syntheticbone surface/synthetic osteoclast” thereby stimulating osteoblastsand/or osteoprecursors to a greater degree than the “unorganized” formsof these factors (i.e., for example, by independent local and/orsystemic administration). See, FIGS. 14A and 14B.

In one embodiment, the present invention contemplates a method of makinga functional artificial osteoblast. In one embodiment, the presentinvention contemplates a method of making a function artificialosteoclast. In one embodiment, the method comprises emulsifying apolymer with a soluble signaling factor to produce a controlled releasemicroparticle. In one embodiment, the controlled release microparticlecomprises surface bound signaling factors. In one embodiment, anartificial osteoblast microparticle comprises a soluble MSCF and surfacebound factors comprising RANK-L, αOSCAR, and/or EPF. See, FIGS. 14A and14B. In one embodiment, an artificial osteoclast microparticle comprisesa soluble MIM1 and surface bound factors EphB4 and/or TGF-β. See, FIGS.15A and 15B. Although it is not necessary to understand the mechanism ofan invention, it is believed that the encapsulation and release of theseagents, and even the ability to precisely pre-program the profile ofrelease maybe measured and/or predicted using mathematical modeling ofcontrolled delivery and variation of the microstructure and chemicalstructure of the particles.

In one embodiment, the present invention contemplates a method forexamining combinations of soluble factors (i.e., for example secreted)and surface-bound factors (i.e.,

for example, immobilized) in vitro using known differentiation markersand functional assays for osteoblast and osteoclast stimulation. In oneembodiment, the method measures the cellular effects via standardco-culture assays. Takahashi et al., Endocrinology 123: 2600-2601(1988); and Suda et al., Endocrinology 128:1792-1796 (1991). In oneembodiment, the method uses assays for osteoblast activity andosteoprecursor differentiation including staining for alkalinephosphatase, Von-Kossa, and flow cytometric analysis. In one embodiment,the method uses assays for calcium phosphate pit-formation and TRAPstaining.

In one embodiment, the present invention contemplates a method for invivo osteo-coupling using artificial osteoblast and/or osteoclast cells.In one embodiment, the method utilizes a subcutaneous, mousecraniofacial model. In one embodiment, the model determines the effectsof combinations of multiple soluble and surface-bound factors byintradermal and/or subcutaneous injections of artificial osteoblastand/or osteoclast cells can be made right under the skin, wherein thecells result in contacting a bone surface. Although it is not necessaryto understand the mechanism of an invention, it is believed that theseartificial cells can be made large enough as to not move from the siteof injection. In one embodiment, the effect of these various artificialcell formulations in vivo are determined using software capable ofquantifying bone density and lacunae (resorption) formation inhistological sections of the craniofacial bone tissue (BioQuant,Osteoimage).

2. Tissue Healing

Periodontal disease, or periodontitis, is characterized by destructiveinflammation of the periodontium (i.e., for example, gum tissue,supporting bone, and/or ligaments) and is considered the most pressingoral health concern today. Importantly, this disease affects not onlytooth loss, but also the incidence of cardiovascular disease, kidneydisease, respiratory diseases, diabetes, and even premature childbirth.Seymour, G. J., Ford, P. J., Cullinan, M. P., Leishman, S. & Yamazaki,K. Relationship between periodontal infections and systemic disease.Clin Microbiol Infect 13 Suppl 4, 3-10 (2007); Fisher, M. A. et al.Periodontal disease and other nontraditional risk factors for CKD. Am JKidney Dis 51, 45-52 (2008); Boggess, K. A., Beck, J. D., Murtha, A. P.,Moss, K. & Offenbacher, S. Maternal periodontal disease in earlypregnancy and risk for a small-for-gestational-age infant. Am J ObstetGynecol 194, 1316-1322 (2006); Backes, J. M., Howard, P. A. & Moriarty,P. M. Role of C-reactive protein in cardiovascular disease. AnnPharmacother 38, 110-118 (2004); and Offenbacher, S. & Beck, J. D. Aperspective on the potential cardioprotective benefits of periodontaltherapy. Am Heart J 149, 950-954 (2005). Periodontal disease isstrikingly prevalent in the United States, affecting 34% of individualsover the age of 30, or an estimated 78 million Americans. Garlet, G. P.et al. Regulatory T cells attenuate experimental periodontitisprogression in mice. Journal of Clinical Periodontology 37, 591-600. Itis the number-one cause of tooth loss according to the American DentalAssociation. Beyond the US, periodontal disease is estimated to affectup to 20% of the adult population worldwide. Furthermore, theperiodontal biofilm hosts a wide variety of potentially hazardousbacterial species that can lead to systemic infections and inflammatoryimmune diseases. The most predominant periodontal pathogens,Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis,Tannerella forsythia and Fusobacterium nucleatum, present virulencefactors that have been associated with: 1) systemic infections andcomplications; 2) a 4-fold increase in premature births, 3)anorexia-cachexia syndrome, 4) atherosclerosis, and 5) mycordialinfarction and ischemic stroke. As the correlations betweenperiodontitis and the incidence of these other conditions continue to beelucidated, reducing the prevalence of periodontal disease is asignificant current medical problem and must be solved. The continuingprevalence of periodontal disease is likely perpetuated by amisunderstanding of the disease etiology consonant with currenttherapies that are aimed at removal of these remarkably common bacterialspecies. The current standard of care involves the debridement ofcalculus and plaque, often accompanied by local delivery of anantibiotic such as minocycline (Arestin®). These conventional treatmentstemporarily kill pathogens, but do not protect against inevitable futureinfections nor address the sensitivity that is observed in patients thatare disposed to immune dysfunction. Although invasive bacterial speciesare protagonists of the disease, tissue destruction is mediated by anadverse host inflammatory immune response. Baker, P. J. The role ofimmune responses in bone loss during periodontal disease. Microbes andInfection 2, 1181-1192 (2000); Graves, D. T., Fine, D., Teng, Y. T., VanDyke, T. E. & Hajishengallis, G. The use of rodent models to investigatehost-bacteria interactions related to periodontal diseases. J ClinPeriodontol 35, 89-105 (2008).

In some embodiments, the present invention contemplates a method fortreating inflammation caused by periodontal disease. Although it is notnecessary to understand the mechanism of an invention, it is believedthat as the disease progresses, several populations of lymphocytes arerecruited to the periodontium, guided by local gradients of specificlymphocyte-attracting chemokines. It is further believed that theoverall cytokine milieu produced by these specific populations oflymphocytes that ultimately directs the expression of factors thatpromote hard and soft tissue destruction.

For example, in some embodiments an artificial antigen presenting cellprovides a therapy for inflammation resulting from periodontal gumdisease. Periodontal disease is strikingly prevalent in the UnitedStates (34% of individuals over age 30 or an estimated 78 millionAmericans). Eke et al., “CDC Periodontal Disease Surveillance Project:background, objectives, and progress report” J Periodontol 78:1366-1371(2007). Periodontal disease is a cause of tooth loss according to theAmerican Dental Association and is a current oral health concern. It isbelieved that periodontal disease not only affects tooth loss but alsocontributes to the incidence of cardiovascular disease, diabetes, andrespiratory disease such as pneumonial. Furthermore, women who haveperiodontal disease are over 4 times more likely to give birth to achild prematurely. Boggess et al., “Maternal periodontal disease inearly pregnancy and risk for a small-for-gestational-age infant” Am JObstet Gynecol 194:1316-1322 (2006). Clearly, finding a solution to theproblem of periodontal disease is needed in the art. In: PennsylvaniaDepartment of Health report: “Status of Oral health in Pennsylvania”(2002).

The physiological events associated with periodontal disease are wellcharacterized but the pathologic mechanisms of this disease are unknown.It is believed that periodontal disease affects the composition andintegrity of periodontal structures at the dento-gingival junction,alveolar bone, cementum and periodontal ligament. Further it is believedthat periodontal disease causes the destruction of connective tissuematrix and cells, loss of fibrous attachment, and resorption of alveolarbone. FIG. 11. Although it is not necessary to understand the mechanismof an invention, it is believed that tissue destruction in periodontalpatients is thought to be a result of a complex inflammatory and immuneresponse initiated and perpetuated by gram-negative anaerobic rods andspirochetes. Previous work has suggested that B cells and T cellsaccumulate in large numbers in the periodontal tissues although muchabout their functions in the disease process is not clearly understood.

Current therapies for periodontal disease focus upon reducing orcontrolling bacterial infection in the gums. Traditional, mechanicaldebridement techniques temporarily remove the accumulated bacterialplaque responsible for inflammation. One treatment strategy provides acontrolled release of the antibiotic minicycline from PLGA microspheresthat are placed in the periodontal pocket (Arestin®, OraPharma).Although both of these techniques may temporarily reduce inflammationassociated with bacteria, they do not intend to treat the susceptibilityof the area to inflammation and the reformation of pockets thatcultivate further infection leading to re-establishment of disease.Since periodontal tissue destruction is initiated and exacerbated byinflammatory host response, there has been an increased focus towardunderstanding the cellular immune responses in the periodontal space inorder to treat the source of the physiological response.

Recently, several groups have discovered evidence that the cause ofperiodontal disease may related to the regulation of inflammation in thelocal tissues. Cardoso et al., “Characterization of CD4+CD25+ naturalregulatory T cells in the inflammatory infiltrate of human chronicperiodontitis” J Leukoc Biol (2008); and Ernst et al., “Diminishedforkhead box P3/CD25 double-positive T regulatory cells are associatedwith the increased nuclear factor-kappaB ligand (RANK-L+) T cells inbone resorption lesion of periodontal disease” Clin Exp Immunol148:271-280 (2007). It was reported that cytokines which may inhibitand/or regulate inflammation (i.e., for example, IL10) are substantiallydiminished in tissues with periodontal disease. Although it is notnecessary to understand the mechanism of an invention, it is believedthat the absence of these anti-inflammatory cytokines not only result inincreased inflammation, but also produces a cascade leading to theproduction of RANK-L. Although it is not necessary to understand themechanism of an invention, it is believed that RANK-L comprises a factorthat differentiates monocyte precursors (i.e., for example, osteoblasts)into bone-resorbing cells called osteoclasts.

The regulation of anti-inflammatory cytokines and more generally, theregulation of the resultant harmful autoimmune responses, may bemediated by immunosuppressive T-cells (i.e., for example, T_(reg)cells). Tang et al., “The Foxp3+ regulatory T cell: a jack of alltrades, master of regulation” Nat Immunol 9:239-244 (2008). For example,it has been reported that while markers for T_(reg) cells were presentin healthy tissues, they were absent in diseased periodontal tissues.Further, it has been reported that patients with rheumatoid arthritis(predisposition for autoimmune regulation breakdown) have 8-foldincreased odds of developing periodontal disease. Pischon et al.,“Association among rheumatoid arthritis, oral hygiene, andperiodontitis” J Periodontol 79:979-986 (2008).

As discussed above, periodontal disease is currently treated withstrategies focused only on the removal of invasive bacterial species.Specifically, the clinical procedure called scaling and root planinginvolves the mechanical removal of plaque and bacteria from beneath thegingiva (i.e., for example, debridement), and is typically preformed bya periodontal specialist. In severe cases, antibiotic treatments in theform of controlled release microparticles may be injected into theperiodontal pocket (i.e., for example, Arrestin®, PLGA microparticlesencapsulating and controllably releasing the antibiotic minocycline for21 days). Williams, R. C. et al. Treatment of periodontitis by localadministration of minocycline microspheres: A controlled trial. Journalof Periodontology 72, 1535-1544 (2001). This treatment approach isinsufficient because, antibiotic treatments only temporarily removes thebacterial species and recurrent infections are common, requiringpatients to repetitively undergo these expensive procedures. While suchantibiotic treatments temporarily remove the bacterial insult, they donot address the sensitivity seen in patients that are susceptible tosuch immune-mediated tissue destruction. Furthermore, antibiotictreatment and/or scaling is completely ineffective in approximately 20%of the population and may induce antibiotic resistance, thereby limitingpatient options for future treatment.

Recent periodontal disease research has investigated the inflammatoryimmune reaction itself. For example, one recent experimental treatmentaimed at reducing the host inflammatory response involves theadministration of the drug Resolvin®. Resolvin® blocksneutrophil-mediated inflammation and the associated pro-inflammatorycytokine milieu. Hasturk, H. et al. Resolvin E1 regulates inflammationat the cellular and tissue level and restores tissue homeostasis invivo. J Immunol 179, 7021-7029 (2007). However, it has been shown thatdirect, long-term, inhibition of inflammatory cytokines by traditionalblocking strategies (i.e., for example, anti-inflammatory compounds) cancompromise periodontal tissue healing. Gurgel, B. C. et al. SelectiveCOX-2 inhibitor reduces bone healing in bone defects. Brazilian oralresearch 19, 312-316 (2005); Ribeiro, F. V. et al. Selectivecyclooxygenase-2 inhibitor may impair bone healing around titaniumimplants in rats. Journal of Periodontology 77, 1731-1735 (2006); Simon,A. M., Manigrasso, M. B. & O'Connor, J. P. Cyclo-oxygenase 2 function isessential for bone fracture healing. Journal of Bone and MineralResearch 17, 963-976 (2002); Vuolteenaho, K., Moilanen, T. & Moilanen,E. Non-steroidal anti-inflammatory drugs, cyclooxygenase-2 and the bonehealing process. Basic and Clinical Pharmacology and Toxicology 102,10-14 (2008); Zhang, X., Kohli, M., Zhou, Q., Graves, D. T. & Amar, S.Short- and long-term effects of IL-1 and TNF antagonists on periodontalwound healing. Journal of Immunology 173, 3514-3523 (2004). Further, ithas also been shown that traditional blocking of the protective immuneresponses actually results in increased bacterial burden and acutesystemic reactions. Garlet, G. P. et al. The essential role of IFN-gammain the control of lethal Aggregatibacter actinomycetemcomitans infectionin mice. Microbes Infect 10, 489-496 (2008); and Garlet, G. P. et al.The dual role of p55 tumour necrosis factor-α receptor in Actinobacillusactinomycetemcomitans-induced experimental periodontitis: Hostprotection and tissue destruction. Clinical and Experimental Immunology147, 128-138 (2007). Consequently, an untested strategy toward diseaseresolution may not require complete blocking of the protective immuneresponses and would clearly be a more desirable approach.

Notably, harmful inflammatory responses are not regulated by blockingleukocyte infiltration, but rather by balancing inflammatory leukocyterecruitment with regulatory lymphocyte recruitment. Vignali, D. A. A.,Collison, L. W. & Workman, C. J. How regulatory T cells work. NatureReviews Immunology 8, 523-532 (2008); and Campanelli, A. P. et al.CD4+CD25+ T cells in skin lesions of patients with cutaneousleishmaniasis exhibit phenotypic and functional characteristics ofnatural regulatory T cells. J Infect Dis 193, 1313-1322 (2006).Regulatory T-cells (Treg) may exert their control over other lymphocytesboth by several mechanisms including but not limited to secretingfactors and through direct cell-cell interactions, ultimately leading totargeted inflammatory-immune cell arrest. However, progress has beenmade toward utilizing Treg therapeutically for a wide variety ofautoimmune and inflammatory diseases. Adoptive (Treg) cell transfer(i.e. cell infusion therapies) have seen the most pre-clinical success.Yet clinical translation of the complicated ex vivo cellular expansionprotocols has proven difficult. Riley, J. L., June, C. H. & Blazar, B.R. Human T Regulatory Cell Therapy: Take a Billion or So and Call Me inthe Morning. Immunity 30, 656-665 (2009). One may spectulate thatperiodontal disease treatment would have the best clinical impact withan off-the-shelf therapeutic that improves the body's natural mechanismsfor immune regulation (recruitment/activation of endogenous Treg) inattempt to restore local immune homeostasis and balance.

Naturally, immune cells are recruited to peripheral sites via chemokinessecreted by tissue resident cells. Specifically, biological gradients ofchemokines direct immune cells toward the origin of secretion (i.e., forexample, at an infection site). For example, one way in which tumorsappear to avoid immune surveillance and clearance is through therecruitment of regulatory T cells. Specifically, tumors produce andsustain a biological gradient of CCL22 (a Treg-associated chemokine)that directs Treg migration. Dutzan, N., Gamonal, J., Silva, A., Sanz,M. & Vernal, R. Over-expression of forkhead box P3 and its associationwith receptor activator of nuclear factor-κ B ligand, interleukin(IL)-17, IL-10 and transforming growth factor-β during the progressionof chronic periodontitis. Journal of Clinical Periodontology 36, 396-403(2009); Sather, B. D. et al. Altering the distribution of Foxp3+regulatory T cells results in tissue-specific inflammatory disease.Journal of Experimental Medicine 204, 1335-1347 (2007); Garlet, G. P.,Avila-Campos, M. J., Milanezi, C. M., Ferreira, B. R. & Silva, J. S.Actinobacillus actinomycetemcomitans-induced periodontal disease inmice: patterns of cytokine, chemokine, and chemokine receptor expressionand leukocyte migration. Microbes Infect 7, 738-747 (2005). Onceco-localized, Treg suppress effector immune cells by secreting factorssuch as IL-10 and TGF-β, thereby establishing immunological homeostasisin an milieu that would otherwise present itself as highly inflammatory.Rabinovich, G. A., Gabrilovich, D. & Sotomayor, E. M. in Annual Reviewof Immunology, Vol. 25 267-296 (2007).

Although it is not necessary to understand the mechanism of aninvention, it is believed that engineering principles may result in theproduction of controlled release systems that can produce a stablebiological gradient of certain chemokines (i.e., for example, CCL22).These microparticles can be fabricated using an FDA-approved polyesterand will degrade in a well characterized manner in vivo. Rothstein, S.N., Federspiel, W. J., Little, S. R. A unified mathematical model forthe prediction of controlled release from surface and bulk erodingpolymer matrices. Biomaterials (2009); Garlet, G. P. et al. The dualrole of p55 tumour necrosis factor-alpha receptor in Actinobacillusactinomycetemcomitans-induced experimental periodontitis: hostprotection and tissue destruction. Clin Exp Immunol 147, 128-138 (2007).Theoretically, by mimicking the natural immune-evasion mechanisms oftumors using rationally-designed, CCL22-releasing, polymericmicroparticles, Tregs may be recruited to a site of destructiveinflammation (i.e., for example, a diseased periodontium).

In one embodiment, the present invention contemplates a methodcomprising a controlled delivery of Treg chemoattractants (i.e., forexample, CCL22 and/or vasoactive intestinal peptide, VIP) in theperiodontium that can abrogate periodontal disease symptoms. In oneembodiment, the method is clinically viable, biocompatible, andnon-inflammatory. Rothstein, S. N., Federspiel, W. J., and Little, S. R.A simple model framework for the prediction of controlled release frombulk eroding polymer matrices. Journal of Materials Chemistry 18,1873-1880 (2008); Rothstein, S. N., Federspiel, W. J., Little, S. R. Aunified mathematical model for the prediction of controlled release fromsurface and bulk eroding polymer matrices. Biomaterials (2009); Delgado,M., Gonzalez-Rey, E. & Ganea, D. VIP/PACAP preferentially attract Th2effectors through differential regulation of chemokine production bydendritic cells. FASEB Journal 18, 1453-1455 (2004); Little, S. R. etal. Poly-beta amino ester-containing microparticles enhance the activityof nonviral genetic vaccines. Proc Natl Acad Sci USA 101, 9534-9539(2004). In one embodiment, the method comprises a controlled releaseformulation that is tunable and provides a long-lasting delivery of adrug. Although it is not necessary to understand the mechanism of aninvention, it is believed that these embodiments provide a potentialsolution for immune imbalance and dysfunction associated withperiodontal disease, while avoiding immune-blocking strategies. It isfurther believed that these embodiments are supported by preliminarydata showing: 1) Tregs are attracted toward a preliminary formulation ofCCL22 microparticles, in vitro and in vivo; 2) administration of CCL22microparticles results in resolution of periodontal disease symptoms inin vivo animal models; 3) transformation/proliferation of CD4+lymphocyte populations into FoxP3+ regulatory lymphocytes is possibleusing factors including, but not limited to, TGF-β, IL2, and/orrapamyacin.

Inflammatory imbalances may cause periodontal tissue destruction whereinthe inflammatory cascade is primarily initiated by bacterial recognitionthrough innate immune cells bearing toll like receptors (TLRs). Garlet,G. P., Avila-Campos, M. J., Milanezi, C. M., Ferreira, B. R. & Silva, J.S. Actinobacillus actinomycetemcomitans-induced periodontal disease inmice: Patterns of cytokine, chemokine, and chemokine receptor expressionand leukocyte migration. Microbes and Infection 7, 738-747 (2005);McGinity, J. W. & O'Donnell, P. B. Preparation of microspheres by thesolvent evaporation technique. Adv Drug Deliv Rev 28, 25-42 (1997);Burns, E., Bachrach, G., Shapira, L. & Nussbaum, G. Cutting edge: TLR2is required for the innate response to Porphyromonas gingivalis:Activation leads to bacterial persistence and TLR2 deficiency attenuatesinduced alveolar bone resorption. Journal of Immunology 177, 8296-8300(2006); Kajita, K. et al. Quantitative messenger RNA expression ofToll-like receptors and interferon-alpha1 in gingivitis andperiodontitis. Oral Microbiol Immunol 22, 398-402 (2007); Nakamura, H.et al. Lack of Toll-like receptor 4 decreases lipopolysaccharide-inducedbone resorption in C3H/HeJ mice in vivo. Oral Microbiology andImmunology 23, 190-195 (2008). Triggering of these pathogen receptorsmay lead to the induction of pro-inflammatory mediators including, butnot limited to, TNF-α, IL-1β and IFN-Y. Garlet, G. P., Martins Jr, W.,Ferreira, B. R., Milanezi, C. M. & Silva, J. S. Patterns of chemokinesand chemokine receptors expression in different forms of humanperiodontal disease. Journal of Periodontal research 38, 210-217 (2003);Garlet, G. P. et al. Cytokine pattern determines the progression ofexperimental periodontal disease induced by Actinobacillusactinomycetemcomitans through the modulation of MMPs, RANKL, and theirphysiological inhibitors. Oral Microbiology and Immunology 21, 12-20(2006). This skewed, pro-inflammatory environment may upregulate softtissue destroying matrix metalloproteinases (MMPs) and/or RANKL areceptor activator of nuclear factor κB ligand and a potent activator ofbone resorbing osteoclasts. Garlet, G. P. et al. The essential role ofIFN-gamma in the control of lethal Aggregatibacter actinomycetemcomitansinfection in mice. Microbes Infect 10, 489-496 (2008); Garlet, G. P. etal. The dual role of p55 tumour necrosis factor-α receptor inActinobacillus actinomycetemcomitans-induced experimental periodontitis:Host protection and tissue destruction. Clinical and ExperimentalImmunology 147, 128-138 (2007); Tang, Q. & Bluestone, J. A. The Foxp3+regulatory T cell: A jack of all trades, master of regulation. NatureImmunology 9, 239-244 (2008); Normenmacher, C. et al. DNA fromperiodontopathogenic bacteria is immunostimulatory for mouse and humanimmune cells. Infection and Immunity 71, 850-856 (2003); Graves, D. T. &Cochran, D. The contribution of interleukin-1 and tumor necrosis factorto periodontal tissue destruction. J Periodontol 74, 391-401 (2003).Inflammatory mediators may result in tissue destruction in theperiodontium and inhibit the production of anti-inflammatory factorsconducive for disease amelioration and healing. Consequently, thepresent invention contemplates a method for solving the periodontaldisease problem by controlling the inflammatory response associated withthe known immunological imbalance associated with periodontitis.

Tregs have been shown to reestablish immune homeostasis through a widevariety of mechanisms, both at the site of inflammation (i.e., forexample, the periodontium) and/or at the draining lymph nodes (i.e., forexample, the cervical lymph nodes). Taubman, M. A., Valverde, P., Han,X. & Kawai, T. Immune response: the key to bone resorption inperiodontal disease. J Periodontol 76, 2033-2041 (2005) Specifically,Tregs act to balance these pro-inflammatory mediators by secretinganti-inflammatory factors including, but not limited to, IL-10 and/orTGF-β. Vignali, D. A. A., Collison, L. W. & Workman, C. J. Howregulatory T cells work. Nature Reviews Immunology 8, 523-532 (2008);Cardoso, C. R. et al. Characterization of CD4+CD25+ natural regulatory Tcells in the inflammatory infiltrate of human chronic periodontitis.Journal of Leukocyte Biology 84, 311-318 (2008). Indeed, recent reportshave shown that IL-10 levels are substantially diminished in patientswith severe periodontitis. Zhang, N. et al. Regulatory T CellsSequentially Migrate from Inflamed Tissues to Draining Lymph Nodes toSuppress the Alloimmune Response. Immunity 30, 458-469 (2009). IL-10 hasbeen shown to play a major role in attenuation of periodontal disease byupregulating an extracellular RANKL inhibitor osteoprotegerin (OPG) andpromoting increased levels of intra-inflammatory-cell ‘suppressors ofcytokine signaling’ (SOCS). Claudino, M. et al. The broad effects of thefunctional IL-10 promoter-592 polymorphism: Modulation of IL-10, TIMP-3,and OPG expression and their association with periodontal diseaseoutcome. Journal of Leukocyte Biology 84, 1565-1573 (2008); Garlet, G.P., Cardoso, C. R., Campanelli, A. P., Martins Jr, W. & Silva, J. S.Expression of suppressors of cytokine signaling in diseased periodontaltissues: A stop signal for disease progression? Journal of PeriodontalResearch 41, 580-584 (2006). Furthermore, IL-10 not only regulates theinflammatory immune response, but also plays a key role in boneanabolism, leading to maturation of bone forming osteoblasts. Abe, T. etal. Osteoblast differentiation is impaired in SOCS-1-deficient mice. JBone Miner Metab 24, 283-290 (2006); Lorentzon, M., Greenhalgh, C. J.,Mohan, S., Alexander, W. S. & Ohlsson, C. Reduced bone mineral densityin SOCS-2-deficient mice. Pediatr Res 57, 223-226 (2005); Ouyang, X. etal. SOCS-2 interferes with myotube formation and potentiates osteoblastdifferentiation through upregulation of JunB in C2C12 cells. J CellPhysiol 207, 428-436 (2006). Finally, TGF-β has been shown to play animportant role in immune regulation and tissue healing, specificallythrough the recruitment and guidance of periodontal ligament cells.Ernst, C. W. O. et al. Diminished forkhead box P3/CD25 double-positive Tregulatory cells are associated with the increased nuclear factor-κBligand (RANKL+) T cells in bone resorption lesion of periodontaldisease. Clinical and Experimental Immunology 148, 271-280 (2007).

In one embodiment, the present invention contemplates a method forrestoring the immunosupressive regulatory balance in periodontal space,thereby reversing periodontal disease progression. In one embodiment,the population of Treg cells in the periodontal space is increased. Inone embodiment, the amount of immunosupressive cytokines (i.e., forexample, CCL-22) in the periodontal space is increased. Although it isnot necessary to understand the mechanism of an invention, it isbelieved that immunosupressive cytokines activate T_(reg) cells thatactually mediate the immunosuppression.

Treg populations have been identified in periodontal lesions. In oneembodiment, the present invention contemplates a method providing acontrolled release of soluble T_(reg) stimulatory factors (i.e., forexample, CCL-22) from soluble biodegradable microparticles afterinjection into a periodontal space. The data presented hereindemonstrates a sustained release of CCL-22 over a period of 1 month frommicroparticles composed of the FDA-approved polymer, PLGA. FIG. 9A.Although it is not necessary to understand the mechanism of aninvention, it is believed that the released cytokine recruits T_(reg)cells back into the periodontal space, thereby restoring immuneregulation and abrogating periodontal disease progression. See, ExampleXII.

In one embodiment, the present invention contemplates a method for thecontrolled delivery of Treg chemoattractants in the periodontium therebypromoting localization of endogenous Treg and abrogating periodontaldisease symptoms. In one embodiment, the method further comprisesrecruiting endogenous Treg to the periodontium. In one embodiment, theperiodontal disease symptoms are abrogated by reducing inflammation.Although it is not necessary to understand the mechanism of aninvention, it is believed that the this method is supported bypreliminary data demonstrating that: 1) controlled release of CCL22leads to the recruitment of endogenous, Foxp3+ regulatory T-cells to theperiodontium; 2) administration of CCL22 controlled release formulationsleads to both higher numbers of Treg in draining lymph nodes andresolution of periodontal disease symptoms in in vivo animal models; and3) beyond recruitment of Treg, a combination of specific factors havebeen identified that could induce the proliferation and/ortransformation of local CD4+ lymphocytes toward enriched Tregpopulations.

In one embodiment, the present invention contemplates formulationscapable of a long-lasting release of Treg-inducing factors. In oneembodiment, the formulation is used for the treatment of periodontitis.In one embodiment, microparticles comprising CCL22 and/or vasoactiveintestinal peptide (VIP), are constructed of biodegradablepoly(lactic-co-glycolic acid) (PLGA) polymer. Rothstein, S. N.,Federspiel, W. J., and Little, S. R. A simple model framework for theprediction of controlled release from bulk eroding polymer matrices.Journal of Materials Chemistry 18, 1873-1880 (2008). In one embodiment,the PLGA microparticles facilitate Treg recruitment to the periodontium.In one embodiment, the microparticle is a controlled releasemicroparticle.

In one embodiment, the present invention contemplates a methodcomprising a microparticle formulation for treating periodontal disease,wherein the formulation promotes Treg recruitment, thereby reducingalveolar bone loss. Although it is not necessary to understand themechanism of an invention, it is believed that local lymphocytes mayexpand toward an enriched population of Tregs through controlled releaseof a combination of several key molecules. Although it is not necessaryto understand the mechanism of an invention, it is believed thatrecruited Tregs may play a role in periodontal disease abrogation andhost response. For example, CCL22 microparticle formulation and systemicVIP effectively induce Treg migration to periodontal tissues and mayabrogate disease symptoms. In one embodiment, the present inventioncontemplates formulations that induce Treg chemotaxis and therapeuticfunction in the periodontium by monitoring Treg residence and diseasesymptoms. For example, normal mice may be compared to mice deficient inreceptors thought to play a role in chemotactic migration andimmunological function. Further, gene expression levels of Treg markers,inflammatory mediators, soft and hard tissue destroying factors andbiomolecules involved in bone growth to elucidate the mechanisms ofTreg-mediated periodontal disease abrogation may also be assessed.

VII. Antibodies

The present invention provides for the use of antibodies (i.e., forexample, polyclonal or monoclonal). In one embodiment, the presentinvention provides monoclonal antibodies that specifically bind to apolypeptide residing on a T_(reg) cell.

An antibody against a protein of the present invention may be anymonoclonal or polyclonal antibody, as long as it can recognize theprotein. Antibodies can be produced by using a protein of the presentinvention as the antigen according to a conventional antibody orantiserum preparation process.

The present invention contemplates the use of both monoclonal andpolyclonal antibodies. Any suitable method may be used to generate theantibodies used in the methods and compositions of the presentinvention, including but not limited to, those disclosed herein. Forexample, for preparation of a monoclonal antibody, protein, as such, ortogether with a suitable carrier or diluent is administered to an animal(e.g., a mammal) under conditions that permit the production ofantibodies. For enhancing the antibody production capability, completeor incomplete Freund's adjuvant may be administered. Normally, theprotein is administered once every 2 weeks to 6 weeks, in total, about 2times to about 10 times. Animals suitable for use in such methodsinclude, but are not limited to, primates, rabbits, dogs, guinea pigs,mice, rats, sheep, goats, etc.

For preparing monoclonal antibody-producing cells, an individual animalwhose antibody titer has been confirmed (e.g., a mouse) is selected, and2 days to 5 days after the final immunization, its spleen or lymph nodeis harvested and antibody-producing cells contained therein are fusedwith myeloma cells to prepare the desired monoclonal antibody producerhybridoma. Measurement of the antibody titer in antiserum can be carriedout, for example, by reacting the labeled protein, as describedhereinafter and antiserum and then measuring the activity of thelabeling agent bound to the antibody. The cell fusion can be carried outaccording to known methods, for example, the method described by Koehlerand Milstein (Nature 256:495 [1975]). As a fusion promoter, for example,polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.

Examples of myeloma cells include NS-1, P3U1, SP2/0, AP-1 and the like.The proportion of the number of antibody producer cells (spleen cells)and the number of myeloma cells to be used is preferably about 1:1 toabout 20:1. PEG (preferably PEG 1000-PEG 6000) is preferably added inconcentration of about 10% to about 80%. Cell fusion can be carried outefficiently by incubating a mixture of both cells at about 20° C. toabout 40° C., preferably about 30° C. to about 37° C. for about 1 minuteto 10 minutes.

Various methods may be used for screening for a hybridoma producing theantibody (e.g., against a tumor antigen or autoantibody of the presentinvention). For example, where a supernatant of the hybridoma is addedto a solid phase (e.g., microplate) to which antibody is adsorbeddirectly or together with a carrier and then an anti-immunoglobulinantibody (if mouse cells are used in cell fusion, anti-mouseimmunoglobulin antibody is used) or Protein A labeled with a radioactivesubstance or an enzyme is added to detect the monoclonal antibodyagainst the protein bound to the solid phase. Alternately, a supernatantof the hybridoma is added to a solid phase to which ananti-immunoglobulin antibody or Protein A is adsorbed and then theprotein labeled with a radioactive substance or an enzyme is added todetect the monoclonal antibody against the protein bound to the solidphase.

Selection of the monoclonal antibody can be carried out according to anyknown method or its modification. Normally, a medium for animal cells towhich HAT (hypoxanthine, aminopterin, thymidine) are added is employed.Any selection and growth medium can be employed as long as the hybridomacan grow. For example, RPMI 1640 medium containing 1% to 20%, preferably10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetalbovine serum, a serum free medium for cultivation of a hybridoma(SFM-101, Nissui Seiyaku) and the like can be used. Normally, thecultivation is carried out at 20° C. to 40° C., preferably 37° C. forabout 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO2gas. The antibody titer of the supernatant of a hybridoma culture can bemeasured according to the same manner as described above with respect tothe antibody titer of the anti-protein in the antiserum.

Separation and purification of a monoclonal antibody (e.g., against acancer marker of the present invention) can be carried out according tothe same manner as those of conventional polyclonal antibodies such asseparation and purification of immunoglobulins, for example,salting-out, alcoholic precipitation, isoelectric point precipitation,electrophoresis, adsorption and desorption with ion exchangers (e.g.,DEAE), ultracentrifugation, gel filtration, or a specific purificationmethod wherein only an antibody is collected with an active adsorbentsuch as an antigen-binding solid phase, Protein A or Protein G anddissociating the binding to obtain the antibody.

Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen (an antigen against theprotein) and a carrier protein is prepared and an animal is immunized bythe complex according to the same manner as that described with respectto the above monoclonal antibody preparation. A material containing theantibody against is recovered from the immunized animal and the antibodyis separated and purified.

As to the complex of the immunogen and the carrier protein to be usedfor immunization of an animal, any carrier protein and any mixingproportion of the carrier and a hapten can be employed as long as anantibody against the hapten, which is crosslinked on the carrier andused for immunization, is produced efficiently. For example, bovineserum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. maybe coupled to an hapten in a weight ratio of about 0.1 part to about 20parts, preferably, about 1 part to about 5 parts per 1 part of thehapten.

In addition, various condensing agents can be used for coupling of ahapten and a carrier. For example, glutaraldehyde, carbodiimide,maleimide activated ester, activated ester reagents containing thiolgroup or dithiopyridyl group, and the like find use with the presentinvention. The condensation product as such or together with a suitablecarrier or diluent is administered to a site of an animal that permitsthe antibody production. For enhancing the antibody productioncapability, complete or incomplete Freund's adjuvant may beadministered. Normally, the protein is administered once every 2 weeksto 6 weeks, in total, about 3 times to about 10 times.

The polyclonal antibody is recovered from blood, ascites and the like,of an animal immunized by the above method. The antibody titer in theantiserum can be measured according to the same manner as that describedabove with respect to the supernatant of the hybridoma culture.Separation and purification of the antibody can be carried out accordingto the same separation and purification method of immunoglobulin as thatdescribed with respect to the above monoclonal antibody.

The protein used herein as the immunogen is not limited to anyparticular type of immunogen. For example, a protein expressed resultingfrom a virus infection (further including a gene having a nucleotidesequence partly altered) can be used as the immunogen. Further,fragments of the protein may be used. Fragments may be obtained by anymethods including, but not limited to expressing a fragment of the gene,enzymatic processing of the protein, chemical synthesis, and the like.

VIII. Kits

In another embodiment, the present invention contemplates kits for thepractice of the methods of this invention. The kits preferably includeone or more containers containing an artificial antigen presenting cellof this invention. The kit can optionally include a soluble T_(reg)stimulation protein. The kit can optionally include a surface T_(reg)stimulation protein. The kit can optionally include nucleic acids suchas FoxP3. The kit can optionally include chemicals capable offunctionalizing the polymer ends of an artificial antigen presentingcell. The kit can optionally include a pharmaceutically acceptableexcipient and/or a delivery vehicle (e.g., a liposome). The reagents maybe provided suspended in the excipient and/or delivery vehicle or may beprovided as a separate component which can be later combined with theexcipient and/or delivery vehicle. The kit may optionally containadditional therapeutics to be co-administered with an artificial antigenpresenting cell.

The kits may also optionally include appropriate systems (e.g. opaquecontainers) or stabilizers (e.g. antioxidants) to prevent degradation ofthe reagents by light or other adverse conditions.

The kits may optionally include instructional materials containingdirections (i.e., protocols) providing for the use of the reagents inthe induction and maintenance of tissue tolerance. While theinstructional materials typically comprise written or printed materialsthey are not limited to such. Any medium capable of storing suchinstructions and communicating them to an end user is contemplated bythis invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

IX. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions(e.g., comprising the artificial antigen presenting cells describedabove). The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary (e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Dosing is dependent on type and amount of tissue that is transplanted.The course of treatment lasting from several days to several months, orin some cases, for the lifetime of the recipient. Optimal dosingschedules can be calculated from measurements of compound accumulationin the body of the patient. The administering physician can easilydetermine optimum dosages, dosing methodologies and repetition rates.Optimum dosages may vary depending on the relative potency of individualoligonucleotides, and can generally be estimated based on EC_(50s) foundto be effective in in vitro and in vivo animal models or based on theexamples described herein. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly. The treating physician can estimate repetition ratesfor dosing based on measured residence times and concentrations of thedrug in bodily fluids or tissues.

EXPERIMENTAL Example I Emulsification of Regulatory T-Cell ModulatingAgents

Preparing emulsions of water soluble factors using biodegradablepolymers is a commonly used strategy to deliver materials over extendedperiods of time and can make administration of factors with short halflives in vivo a reality. The use of thedouble-emulsion/solvent-evaporation technique is a commonly usedstrategy. Odonnell et al., Advanced Drug Delivery Reviews 28: 25-42(1997), See, FIG. 5. A frequently used polymer in these formulations ispoly-lactic-co-glycolic acid (PLGA), which is both biocompatible and FDAapproved. PLGA is also attractive because its degradation rates can becontrolled by the ratio of its monomers (lactide which is morehydrophobic, and glycolide which is more hydrophilic). This control overdegradation behavior provides the ability to release factors overperiods from days to months. Hanes et al., Pharm Biotechnol 6:389-412(1995). Microparticles prepared in this manner can carry large payloadsand encapsulate multiple agents including, but not limited to: i)cytokines (Thomas et al., J Pharm Sci 93: 1100-1109 (2004)); ii) smallpeptides (Haining et al., J Immunol 173:2578-2585 (2004)); iii) smallmolecule drugs; and iv) nucleic acids (Little et al., Proc Natl Acad SciUSA 101: 9534-9539 (2004)). The size can be controlled by parameterssuch as the concentration of the polymer solution, agitation speedsduring fabrication, and amount of surfactant used in the outer aqueousphase during the emulsification procedure. These formulations can beadministered in a variety of ways, including injection through a needleand syringe.

Any number of compounds may be emulsified using the above methodology.However, of most interest to the embodiments discussed herein, factorswhich are thought to influence the anti-inflammatory environment duringtolerance induction are completely compatible with the system describedhere (i.e., for example, IL2, TGF-β, and CCL22). Further, theemulsification and release of IL2 in PLGA microparticles using a doubleand single emulsion systems has recently been reported. Thomas et al., JPharm Sci 93: 1100-1109 (2004). Specifically, IL2 was encapsulated andreleased in an active state for periods of up to 4 months. Similarly,TGF-β has been encapsulated in microparticles using PLGA polymer blendswith release demonstrated for a period of over 1 month. Kempen et al., JBiomed Mater Res A 70:293-302 (2004); and Lu et al., J Biomed Mater Res50:440-51 (2000). CCL20, but not CCL22, has been encapsulated in PLGAmicroparticles which have been shown stimulate significant amounts of invitro dendritic cell chemotaxis (i.e., for example, over 0.5 mm fromtheir original position to the point of contact with the particle). Zhaoet al., Biomaterials 26:5048-5063 (2005). Importantly, this chemotaxiswas greater than that following bolus administration of the chemokine.CCL22 has been reported to induce regulatory T-cell chemotacticmigration both in vitro and in vivo. Curiel et al., Nat Med 10:942-949(2004).

Example II Labeling Artificial Presenting Cell Surface PolymerFunctionalization

This example provides a simple, rapid detection method to detectparticle surface polymer modifications capable of attaching to solubleand membrane T_(reg) stimulation factors.

Particle surface labeling of biodegradable polymers has beenaccomplished using several established techniques. The most commonstrategy for PLGA is to utilize carboxylic acid groups, which arepresent at the end groups and are generated upon degradation of theester bonds in its backbone. This can be accomplished using carbodiimidechemistry and can be facilitated using NHS esters. See, FIG. 6.

Another approach has been to create more of these carboxylic acid groupson the surface using a surfactant which is rich in these chemical groupsduring fabrication of the particle. Keegan et al., Macromolecules37:9779-9784 (2004). Also, short treatments with 0.1M NaOH can generatean abundance of these groups on the particle surface.

A new technique is presented in this example that quantifies surfacelabeling using a modification of the carbodiimide chemistry approach. Inthis technique, 2-pyridyldithioethylamine, reacts with activated estersand also contains a disulfide bond which is, in turn, readily reactedwith thiol compounds. See, FIG. 7 middle panel. This reaction liberatesa new chemical entity which absorbs light at 342 nm. See, FIG. 7 rightpanel.

Light absorption can be readily detected using any standard absorbanceplate reader. The absorbance data, along with the total amount ofparticles and their average surface area, can determine an estimate ofthe number of labeling events per unit area on the particle surface.This technique has successfully labeled the surface of 10 micrometerPLGA microparticles wherein dithiothreitol was used to liberate theabsorbent product (data not shown).

Alternatively, streptavidin may be used to facilitate the binding ofcommercially available biotin-labeled antibodies. For example,EDC/carbodiimide and NHS chemistry can take place in MES buffer in whichmicroparticles are suspended. The reaction tube will need to beconstantly rotated to keep particles evenly suspended for approximately2 hours. Particles will then be washed and 2-pyridyldithioethylamine(PDA) will be added to the activated ester by 4 hour incubation underagitation.

After washing, these particles are labeled with thiolated streptavidinin excess. Particles will be spun down and supernatants removed foranalysis at 343 nm.

Values obtained will be compared to a standard curve to determine thenumber of moles reacted/weight of particles. The average total availablesurface area for reaction can be determined by preparing an aqueoussuspension of particles with a known concentration. This suspension willbe analyzed by Coulter Counter to determine the number of particles perunit weight. The value, along with the average diameter of the particlesand the number of moles of streptavidin reacted/weight particles leadsto the number of streptavidin linkages per unit area on themicroparticle surface. This value can be checked with the use ofcommercially available biotin conjugated to a fluorescent linker.Particles could be labeled with this material, washed, dissolved usingNaOH/SDS, and this solution's fluorescence could be measured. Thisintensity, as compared to a standard curve, would indicate the amount oflabeling/weight of particles. A pH insensitive fluorescent probe wouldneed to be used in these experiments. These fluorescently labeledparticles will also be incubated (n=3) in a similar fashion as describedabove for the controlled release assay. Stability of the surface linkagewill be determined by the amount of fluorescence in the supernatantafter centrifugation (label that has been liberated from the particlesurface)

Example III Emulsification of Soluble T_(reg) Cell Factors in PLGAMicroparticles

This example describes the emulsification (i.e., for example,encapsulation) of soluble factors within PLGA microparticles using amodified double emulsion procedure. Odonnell et al., Advanced DrugDelivery Reviews 28:25-42 (1997).

Briefly, 20 μg of protein factor and 20 mg of bovine serum albumen willbe dissolved in 200 μL of sterile PBS and added to a solution of 100 mgof 10, 18-30, or 40-78 kDa PLGA with a lactide:glycolide monomer ratioof 50:50 in 2 ml of dichloromethane. This two phase system will besonicated for 10 seconds using a probe sonicator to form the primaryemulsion. The emulsion will immediately be transferred to 50 mL of ahomogenizing (5000 rpm), aqueous solution containing 5% by weightpoly(vinyl) alcohol (PVA) which serves as a surfactant. After 30 secondsof homogenization, the double emulsion is transferred to a stirringsolution of 1% PVA (100 mL) to allow for evaporation of thedichloromethane over a period of 3 hours. The particles will then becentrifuged at <150 rcf in a refrigerated centrifuge and the supernatantcontaining PVA will be decanted. Particles will be resuspended, washedwith cold water, and the process repeated 3×. Washed particles will thenbe suspended in a minimal amount of water and lyophilized for 3 days toallow sufficient time for removal of residual chloroform. Smallerparticles can be prepared using homogenization at 7000 rpm (1 μm).Larger particles can be prepared using a lower concentration ofsurfactant in the outer aqueous phase of the emulsions (i.e. PVA=1% and0.5% by weight) which will result in 10-20 μm particles.

Particle size will be determined by a Coulter Counter using volumeimpedance. Particle loading will be checked by exposing 5 mg ofparticles to a 0.5 M NaOH, 2% SDS solution to promote total degradationof the particles and dissolution of the protein. Concentrations can bedetermined using a BCA assay kit via the manufacturer's instructions andloading can be determined (n=3). It should be reasonable to assume thatthe relative amount of T_(reg) cell factor will remain the same withrespect to BSA during encapsulation. However, this assumption can bechecked using factor-specific ELISA in place of the BCA assay initially.A typical encapsulation efficiency using thedouble-emulsion/solvent-evaporation method for proteins is approximately50%.

Example IV Controlled Release of Soluble Factors from PLGAMicroparticles

This example provides a method to determine the controlled release ofsoluble factors from microparticles made in accordance with Example III.

Approximately 10 mg of microparticles (n=3) will be weighed out intomicrocentrifuge tubes and suspended in 500 μL of PBS. Tubes will besealed and placed on a shaker plate at 37° C. At 12 hour time intervals,tubes will be centrifuged, supernatants removed and stored at −70° C.,and new buffer will be added to the particles. At the end of 2 weeks,supernatants will be analyzed for T_(reg) cell stimulating factor usinga specific ELISA and release will be plotted over time with standarddeviations. Values obtained for all particle formulations in thesestudies can be used for setting up the appropriate conditions formeasuring the activity of these factors and determining their effects onT_(reg) cells in vitro.

Example V Primary Regulatory T-Cell Isolation

Lymph nodes will be harvested from BALB/c mice and processed to achievea single cell suspension. Magnetic cell sorting (MACS) will be employedto purify the population via a depletion step which enriches the CD4+population of cells (mostly CD4+CD25−) and a positive selection stepinvolving CD25 (resulting in mostly CD4+CD25+). Cells will be culturedthereafter in RPMI 1640 media including 10% FCS, glutamine, HEPES,nonessential amino acids, penicillin/streptomycin, and2-mercaptoethanol.

Example VI Biological Activity of Encapsulated TGF-β and IL2

For the determination of TGF-β and IL2 activity, the effect of releasedfactors are measured on a population of CD4+ cells prior to positiveselection of CD25. This assay is takes advantage of the fact that TGF-βand IL2 can aid in the differentiation of peripheral CD4+ cells toincrease their expression of FoxP3 and suppress alloreactivelymphocytes. Fu et al., Am J Transplant 4:1614-27 (2004). Both TGF-β andIL2 are present at the same time as TCR engagement thereby facilitatingactivation.

CD4+CD25− cells are treated for 5 days with experimental groups, mAbspecific to CD3 (1 μg/ml), and syngenic, lymphocyte-depleted andirradiated splenocytes. To examine biological activity of encapsulates,the following experimental groups (n=3) are used: 1) fresh, TGF-β andIL2 (1 ng/ml) as a positive control, 2) particles with no encapsulatedfactors and no external addition of IL2 or TGF-β, 3) IL2 particleincubation with the cells+(1 ng/ml fresh TGF-β), 4) TGF-β particleincubation with the cells+(1 ng/ml fresh IL2).

After treatment, cells will be analyzed by flow cytometry for theincreased presence of CD25 and intracellular expression of FoxP3 (viaCytofix/Cytoperm kit and labeled FoxP3 mAb) which would indicateactivation of the cells. The amount of particles to be used in theseexperiments will be based on the results of the controlled release assay(described above) to allow for equivalent amounts of administered growthfactor. Groups 3 and 4 will be compared to Group 1 to determine thebiological activity of the released IL2 and TGF-β, respectively.

Example VII Measurement of T_(reg) Cell In Vitro Chemotaxis

This example provides one method for measuring the in vitro chemotaxisof T_(reg) cells in response to chemokine release (i.e., for example,CCL22) from particles. See, FIG. 8.

Regulatory T-cells will be isolated as described above using greenfluorescent protein transgenic mice (GFP+−C57BL/6 mice). This providesfluorescently labeled cells which have not been treated by any externalreagents. Microparticles containing CCL22 (n=3) will be placed belowopaque, transwell filters (mean pore size=3 μm and 5 μm). Cells will beseeded on top of this membrane and allowed to incubate for 5 hoursbefore measuring fluorescence. A top and bottom fluorescent readingformat is used which allows for the determination of the number offluorescent cells that have migrated through the membrane at any point.No checkerboard analysis is needed to differentiate between chemotaxisand chemokinesis given that the only factor we are using in ourexperiments is a well known chemotactic agent.

Alternatively, a carboxyfluorescein diacetate succinimidyl ester, orCSFE (a factor used to track T-cell proliferation), may be used insteadof the GFP transgenic mice to label the T_(reg) cells. A manual countingof cells both below and above the transwell filter (using unlabeledcells as a control) should be performed to determine if CSFE has anyeffect on the chemotaxis of the regulatory T-cells.

Experimental controls include, but are not limited to: 1) wells withblank microparticles (as a negative control); and 2) wells containingsoluble CCL22 at 0.5-20 ng/ml (the typical manufacturer's stated ED50range for this material) as a positive control. The amount ofmicroparticles used in this experiment are derived from the controlledrelease experiment (described above) to equalize the released CCL22 withthe positive controls.

A t-test using two-tailed, unequal variance will be used to determinedifferences between groups with p<0.05 being deemed significant.

Example VIII In Vitro Measurement of Soluble Released Factors

This example identifies one embodiment for measuring the effect ofsurface co-stimulatory and soluble released factors on T^(reg) cells invitro.

Materials

Latex beads with streptavidin can be easily purchased in 1, 5, and 10 μmsizes and can be labeled in one step with biotinylated mAb specific toCD3 and CD28. Optionally, the surface bound moieties can be optimized.

In addition to determining the optimal size for the surface labeledmicroparticles, label ratios are also varied to optimize theproliferation of primary T_(reg) cells (i.e., for example, 0% CD3/100%CD28; 25% CD3/75% CD28, 50% CD3/50% CD28; 75% CD3/25% CD28:100% CD3/0%CD28).

Furthermore, saturating concentration of the stimulatory factors on theparticle surface may or may not be optimal for T_(reg) cellproliferation. For example, stimulation of regulatory T-cells by CD28requires an optimized dose. Fu et al., Am J Transplant 4:1614-27 (2004);Salomon et al., Immunity 12:431-40 (2000). To determine this dose, thetotal percentage of streptavidin sites accessible to mAb bound biotinare reduced (i.e., for example, by limiting the addition of free biotinduring the labeling procedure). All samples (n=3) will be compared usinga two-tailed t-test (unequal variance) with p<0.05 being deemedsignificant.

Methods

Primary T_(reg) cells will be isolated from C57BL/6 mice and expandedusing helper cell-free in vitro conditions including the addition offresh IL2. The latex bead system will serve as a positive control forcomparison with labeled PLGA microparticles of different sizes. Negativecontrols will include untreated cells and cells treated with particleswithout surface label.

Soluble IL2 will be replenished, and the cells recounted bi-weekly,while new particles will be administered twice over a two week period.Besides measurement of total regulatory T-cell proliferation, sampleswill be taken from the culture flasks bi-weekly for flow cytometryanalysis. Cells will be stained with fluorescently labeled mAbs whichrecognize CD25, and CD4. Intracellular staining for FoxP3 is used todetermine the activation state of the regulatory T-cells.

Labeling both IL2 and IL2+ TGF-β encapsulated particles are performedunder the same conditions which produced the optimal surface labeledformulation. Also, this protocol will allow the determination if anartificial antigen presenting cell is capable of activating and inducingthe proliferation of regulatory T-cells without the need for manualaddition of soluble factors.

Example IX In Vivo Injection of Chemokine Encapsulated Particles

This example demonstrates the injection of chemokine encapsulatedparticles to determine recruitment capacity in vivo.

Particles will be prepared with the full amount of CCL22 (as describedin the first experimental section), ⅓ this amount, and 1/10 of thisamount. Primary cells (1×10⁵) from transgenic GFP+-B6 mice will beadoptively transferred into normal B6 mice for in vivo chemotaxisexperiments. At this time mice will be given an injection of the threetypes of CCL22 encapsulated microparticles in a solution of sterile PBSand 1% carboxymethylcellulose (to avoid particle settling and needleclogging with the high concentrations of particles used). This injectionwill either be on a shaved and sterilized belly of the animal (i.e., forexample, intraperitoneally) (n=3) or in the tibealis anterior muscle(hind leg) (n=3). On the other side of the animal, blank microparticleswill be injected S.C. or I.M. as a control. Another experimental groupprovides an S.C. or I.M. injection to mice using a soluble CCL22 in thesame amount as the CCL22 encapsulated particles (full amountencapsulated) (n=3).

At t=1 day and 3 days, mice will be euthanized and skin or muscle at thesite of an injection will be resected for histology sections. In thesesections, the more GFP expressing cells near the site of the depot, thegreater the success of the formulation. For a more quantitativeanalysis, we will form a single cell suspension with the resected tissueand analyze using flow cytometery (at 488 nm) to determine how manyadoptively transferred regulatory T-cells were recruited compared to theblank microparticle-treated, negative control (deemed statisticallysignificant by t-test, p<0.05).

Example X In Vivo Injection of Artificial Antigen Presenting Cells

This example demonstrates the injection of artificial antigen presentingcells to determine manipulative effect on T_(reg) cells in vivo.

Artificial antigen presenting cells will be prepared by encapsulating anoptimized CCL22 formulation plus IL2 and TGF-β followed by an optimizedsurface labeling strategy. Experimental groups will be designed toexplore the necessity of the included factors for stimulation of therecruited regulatory T-cells in vivo (i.e. for example, one formulationwith each soluble and surface stimulating component being absent). Micewill be adoptively transferred with transgenic, GFP+ regulatory T-cellsas described above and administered injections in the same manner, butthis time with the artificial APC formulations instead of encapsulatedCCL22 alone (n=3 for each group). Cells from resected skin and musclewill be intracellularly stained for the level of FoxP3 expression (giventhe strong correlation between its up-regulation and T_(reg) cellstimulatory capacity).

These experiments are repeated with the intent of sorting CD4+ cellsinstead of intracellular FoxP3 staining. The sorted cells willsubsequently be used as suppressor cells (suppressor: responder ratiosof 1:1, 2:1 and 5:1) with freshly isolated CD4+CD25− which have beenpre-labeled with CSFE (15 μM) and stimulated for 3 days using monoclonalantibodies specific for CD3 (1 μg/ml) and syngenic, irradiatedsplenocytes (depleted of lymphocytes). Cultures will be monitored usingflow cytometry. Efficient suppression would result in a decreaseddilution (or higher signal at 488 nm) of CSFE in the cellular populationdue to the fewer cell divisions of a suppressed lymphocyte. To determinethe necessity of each of the individual factors, experimental groupswill be compared via two tailed t-test assuming unequal variance withp<0.05 being considered significant.

Example XI Induction of Allograft Tolerance Using Artificial AntigenPresenting Cells

This example demonstrates therapeutic use of optimized artificialantigen presenting cells for induction of allograft tolerance.

The results of the in vivo suppressor assay (supra) provides optimizedformulations and are used for the in vivo allograft tolerance assays.These optimized formulations will be co-administered using either adepot administration of, or by coating the transplant in, artificialantigen presenting cells (n=5). Controls will include mice with notreatment and mice administered (15 mg/kg day) cyclosporine S.C. as apositive control (n=5). Furthermore, an optional experimental groupcomprising an artificial APC group with an initial cyclosporine dose of15 mg/kg day, and then reduced to examine the effect ofimmunosuppressant promotion or inhibition of immune tolerance induction(n=5). The experimental endpoint will be 100 days post operation.

For skin transplantation, mice will be shaven, sterilized, andanesthetized before preparation of two graft beds on the posterior chestwall. Rosenberg A. S. (1991), eds. Coligan, J. E., Kruisbeek, A. M.,Margulies, D. H., Shevach, E. M. & Strober, W. (John Wiley and Sons, NewYork), pp. 4.4.1-4.4.12. These graft beds will engraft full thicknessskin grafts from the tail of a donor and syngenic control. After oneweek of recovery, the transplant will be monitored daily for redness,hair growth, hemorrhaging, and status of graft borders. The percentageof viable skin will be recorded daily until only 10% remains, at whichtime the animal will be euthanized.

Heterotopic heart transplants will be performed as previously described.Zhang et al., Transplantation 62:1267-1272 (1996). Rejection will bemonitored via direct abdominal palpitation using the following rankingsystem: A). Strongly beating, B) Noticeable decline from A, or C)Termination of pulsation at which time the animal will be euthanized.

Following termination of the animals in both transplant models, theallografts will be fixed in 10% formaldehyde and sent for embedding,staining, and evaluation of rejection. Percentage of surviving grafts ineach group will be plotted against time for comparison.

A log rank test will be used to compare groups with transplantationrejection data as analyzed by any one of many available softwarepackages.

Example XII Restoring Immunological Regulation in Periodontal DiseasedTissues

Results Overview

In this experiment, artifical antigen presenting cells providing aCCL-22 controlled release formulation were injected into palatalgingival tissue of right molars of mice (n=3) at the times of −1, 10 and20 days of periodontal infection. At 30 days, total RNA was extractedfrom periodontal tissues as previously described and analyzed forregulatory T-cell and periodontal disease factor. Garlet et al., “Theessential role of IFN-gamma in the control of lethal Aggregatibacteractinomycetemcomitans infection in mice. In: Microbes andinfection/Institut Pasteur (2008). Infected, untreated mice (AA) andnon-loaded particle treated (CP) groups had low levels of FoxP3 (aregulatory T-cell marker) and IL10 (a suppressive cytokine secreted byT_(reg) cells), but elevated levels of RANK-L (an osteoclastdifferentiation factor). See, FIGS. 12A & 12B. Conversely, CCL-22particle treatment groups (LP) had significantly higher levels of FoxP3and IL10, and significantly lower levels of RANK-L. To determine if theparticles had therapeutic effect, mice were given the aforementionedtreatments and alveolar bone loss was measured at 30 days as described.Garlet et al., “Actinobacillus l actinomycetemcomitans-inducedperiodontal disease in mice: patterns of cytokine, chemokine, andchemokine receptor expression and leukocyte migration”. Microbes andinfection/Institut Pasteur 7, 738-747 (2005). Mice treated with CCL-22loaded particles (LP) exhibited significantly reduced bone loss comparedto neg. controls. See, FIG. 12B.

Methods

Fabrication and Characterization of Controlled Release Formulations:Encapsulation of recombinant murine CCL-22 into PLGA microparticles wasperformed using a double emulsion procedure to generate particles in the10 μm range (supra). Loading was verified by dissolution (NaOH, DMSO)followed by protein detection assays such as ELISA to detect totalprotein encapsulated. Particle sizing and SEM analysis were performedfor quality control purposes.

Example XIII Therapeutic CCL-22 aAPCs in a Murine Mouse Model forPeriodontal Disease

To induce periodontal disease in a mouse, we delivered 1×10⁹ CFU of adiluted culture of Aggregatibacter (Actinobacillus)actinomycetemcomitans JP2 (anaerobically grown in supplemented agarmedium, TSBV) in 100 μl of PBS with 2% carboxymethylcellulose, placed inthe oral cavity of mice with a micropipette in accordance with ExampleXII. This procedure will be repeated at 48 and 96 hours later to fullyestablish periodontal disease.

Experimental groups will comprise of eight-week-old C57Bl/6 mice. CCL22loaded particles will be injected in the palatal gingival tissue ofright molars from the mesial of first molar until the distal face of thethird molar, at the times of −1, 10 and 20 days of infection. Negativecontrols will include non-infected and sham-infected mice, which receiveheat-killed bacteria in 2% carboxymethylcellulose solution, mice thatreceive PBS injection in left maxilla of the same LP group, and micethat receive the injection of control non-loaded particles. For apositive control, we will utilize mice that receive regular IPinjections of VIP (vasoactive intestinal peptide) as this materialinduces systemic proliferation of regulatory T-cells. Chorny et al.,“Vasoactive intestinal peptide induces regulatory dendritic cells thatprevent acute graft-versus-host disease while maintaining the graftversus-tumor response” Blood 107:3787-3794 (2006). After 30 days ofinfection, mice will be euthanized and the samples collected for thefollowing experimental analyses:

Alveolar Bone Loss/Healing Analysis. Evaluation of the extent ofalveolar bone loss will be performed in accordance with Example XII andFIG. 12 above. Further, quantitative histological methodology will beused to characterize bone healing. Briefly, the maxillae will behemisected, exposed overnight in 3% hydrogen peroxide and mechanicallydefleshed. The palatal faces of the molars will be photographed at 20×magnification using a dissecting microscope (Leica, Wetzlar, Germany),and the images will be analyzed using ImageTool 2.0 software (Universityof Texas Health Science Center, San Antonio). Quantitative analysis willbe used for the measurement of the area between the cement-enameljunction (CEJ) and the alveolar bone crest (ABC) in the 3 posteriorteeth, in arbitrary units of area (AUA). In total, 5 animals will beanalyzed, and for each animal, the alveolar bone loss will be defined asthe average of CEJ-ABC between the right and left arches.

Histological Assessment for Bone Healing. At the specified times,animals will be euthanized and block sections removed forimmunohistochemistry analyses. Tissue sections will be divided into 2groups (every other section), one for histological/histomorphometryanalysis and the other for immunohistochemistry to assess new boneformation by staining for the Dentin Matrix Protein 1 and BoneSialoprotein.

Real-Time PCR Analysis. Extraction of total RNA from periodontal tissues(upper molars with whole surrounding buccal and palatal periodontaltissues) will be performed with Trizol reagent (Invitrogen, Rockville,Md.) and cDNA synthesis. RealTime-PCR quantitative mRNA analyses will bedone in a MiniOpticon system (BioRad, Hercules, Calif.), usingSybrGreenMasterMix (Invitrogen), 100 nM specific primers, and 2.5 ng ofcDNA in each reaction. We will analyze tissues for the presence ofCCL-22, FoxP3, IL-10, RANK-L, and TGF-b. For mRNA analysis, the relativelevel of gene expression is calculated in reference to beta-actin usingthe cycle threshold (Ct) method.

Quantification and Visualization of Regulatory T-cells. Tissue will beresected and either: 1) sectioned for staining specific for regulatoryT-cells using reported methods (Raimondi et al J Immunol 176, 2808-16(2006) or 2) digested and separated using Ficoll in order to isolatewhite blood cells for flow cytometric analysis. The latter will allow usto determine the degree of FoxP3 expression specifically in regulatoryT-cells (directly correlating to suppressive activity and also thepercentage total regulatory T-cells in the periodontal space incomparison to other lyphocyte populations, CD4+CD25−, CD8+.

Statistical analysis. Preliminary data (n=3 mice per group with aninternal control) obtained statistical significance which was slightlyless than the p=0.05 cutoff. Power analysis based upon standarddeviations and response magnitudes in pilot data (assuming power=0.8)indicates that we require n=5 to obtain desired confidence in ourexperimental results. Statistical significance between the infected andcontrol mice of both strains will be analyzed by ANOVA, followed byBonferroni post test. Values of P<0.05 will be considered statisticallysignificant. We will also test both 1 month and 2 month time-points toexamine whether or not therapeutic efficacy can be maintained andwhether or not healing occurs at these time-points. PCR and AUA basedmeasurements of bone loss/healing can be performed on the same set ofmice (n=5×2 timepoints×4 groups) and histological analysis and flowcytometry can be performed on another set of mice (n=5×2 time-points×4groups) leading to a total of 80 mice. Experimental groups include: 1)sham control, 2) blank particle control, 3) VIP positive control, and 4)CCL-22 particle experimental group.

Example XIV Making Anisotropic Microspheres with Soft Protein Islets

Commercially available carboxylated phosphatydlserine (PS) microspheres(mean diameter 6.37 μm) were purchased from Bangs Laboratories Inc, USA.In preparation for the procedure, microspheres were washed twice indeionised water, then washed once in ethanol, centrifuged at 3500 rpmfor 5 min, and re-suspended in water. Microbiology-grade glass coverslips were washed in soap solution, distilled water, and then ethanol(70%) under bath sonication for 15 min each. “Microwells” were developedby spotting 3 μl of microsphere suspension (10% w/v in water) onto acover slip and dried sequentially at 25° C. for 1 hr 40° C. for 30 minand 60° C. for 10 min (Tg of PS bulk is 100° C. and reduces to 70° C. asthe thickness reduces) 27 to avoid surface melting. The wells werefurther filled 4 times with concentrated microsphere suspension (30%w/v) in water with intermittent drying at 4-8° C. between additions. Thecolloidal crystals were further dried at room temperature for 3 hours.Prior to the addition of PDMS, cover slips with colloidal crystals werepreheated to 60° C. for 5 min on a leveled hot plate (The PDMS((PDMS/catalyst) (10:1) (w/w)) (Sylgard 184 silicone elastomer kit,Dowcorning Corp, MI, USA) solution was added and allowed to saturate andstabilize the interstitial spaces for a minute. This mixture was thenimmediately heated to 90° C. for 15 min. After setting of the PDMSscaffold (16 hrs at 40° C.), the cover slip was carefully removed toprepare the PDMS scaffold/colloidal crystal for protein patterning.

For protein labeling, scaffolds were first immersed in 0.1% Tweensolution, to reduce the non-specific adsorption of proteins. Biotin-PEO(EZ Link®, Pierce, USA) was immobilized onto the microspheres in thePDMS scaffold. Carboxylate groups on the surface of microspheres (exceptthe region at PDMS mask) were activated with 0.1M1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Acros, N.J., USA),and 0.2M N-hydroxysulfosuccinimide (Sulfo-NHS) (Thermo scientific, IL,USA) in 200 μl in 0.2M MES (pH5.5) buffer for 2 hours. Following thereaction, microspheres were washed two times with MES buffer prior toaddition of 50 mM biotin-PEO (Fisher scientific, USA) in 200 μl MESbuffer. After 2 hrs, the solution was removed and microspheres werewashed two times with MES buffer in the scaffold. For etching, TetraButyl Ammonium Flouride (TBAF) (1M solution in Tetrahydrofuran,sigma-Aldrich Inc) in 1-Methyl, 2-Pyrrolidininone (NMP) (Sigma-AldrichInc)/Deionised Water (1:6)(v/v) in 0.1% Tween 80 solution was used.After 1 hour incubation of microspheres in etchant solution, thescaffolds were washed two times with the MES buffer. The second proteinwas then immobilized to the newly exposed area using EDC-NHS chemistry.For that the succinimide derivatized particles 200 μl of 0.2 mM albuminrhodamine (Albumin from bovine serum, tetramethyl rhodamine conjugate,Invitrogen) in 0.1% Tween 80/MES buffer (pH 5.5) was added to label thepatches. The reaction proceeded for 2 hrs in the dark followed bywashing with 1% (w/v) Bovine Serum albumin (BSA) (Sigma-Aldrich Inc) in200 μl MES (pH 5.5) buffer. For labeling of the remaining surface of theparticle, 200 μl MES buffer (pH 5.5) with 0.1% Tween and 1% BSAcontaining 0.2 mM avidin flourescein (Immunopure®, Pierce, USA) wasadded to the washed microspheres in the scaffolds. After 2 hours, thescaffolds were washed two times with MES buffer (pH5.5), and furtherwashed one time in deionised water prior to storage at 4-8° C. Themicrospheres were scrapped out of the scaffold with a steel spatula.Optical microscopy images were taken with an Optical microscope (Caltexsystems 3D Digital Video Inspection measurement system+signatone 1160Probe Station). SEM studies were done with JSM 6330F. CLSM studies wereperformed using an Olympus FluoroView 1000 confocal microscope.

Example XV Microparticle Fabrication

Rationally design of controlled release microparticles may be createdusing a predictive mathematical model. S. R. Little, S. N. Rothstein, W.J. Federspiel, Journal of Materials Chemistry 18, 1873 (2008); S. N.Rothstein, Federspiel, W. J., Little, S. R., Biomaterials, (2009). Themodel allows computationally design formulations with specific releaseprofiles based on drug/polymer characteristics and the desired releasekinetics. Computer generated fabrication parameters are then used toinform the production process. Specifically, emulsion technique canencapsulate drugs in microparticles. FIG. 21. This model specifies thatformulations should have the following characteristics to produce a 20day linear release profile: Particle Size=20 μm; Internal Drug PocketSize=500 nm; Average Polymer Molecular Weight=15 kDa;Polydispersity=1.5. FIG. 16.

Furthermore, a VIP (vasoactive intestinal peptide) microparticleformulation will be designed to provide a constant supply of the peptideover 20 days using the following characteristics: Particle Size=20 μm;Internal Drug Pocket Size=300 nm; Average Polymer Molecular Weight=15kDa; Polydispersity=1.5. If VIP release behavior deviates from thedesigned profile, we are able to easily reformulate the microparticlesto appropriately address the deviant characteristic. Release of VIP frommicropariticles are expected to provide data similar to FIG. 16.

I claim:
 1. A synthetic microparticle comprising at least one polymerand an internal structure, said internal structure comprising aplurality of compartments, wherein a soluble CCL22 chemokine isencapsulated within said plurality of compartments.
 2. The syntheticmicroparticle of claim 1, wherein said microparticle is biodegradable.3. The synthetic microparticle of claim 2, wherein said biodegradablemicroparticle undergoes a controlled chemokine release.
 4. The syntheticmicroparticle of claim 3, wherein said controlled chemokine release is ashort term release comprising between ten-twenty four hours.
 5. Thesynthetic microparticle of claim 3, wherein said controlled chemokinerelease is a long term release comprising between twenty four hours andone year.
 6. The synthetic microparticle of claim 3, wherein saidcontrolled chemokine release is maintained for at least twenty days. 7.The synthetic microparticle of claim 1, wherein said at least onepolymer comprises a polylactide-co-glycolide polymer.
 8. The syntheticmicroparticle of claim 1, wherein said at least one polymer comprises alactic acid monomer.
 9. The synthetic microparticle of claim 8, whereinsaid at least one polymer further comprises a glycolic acid monomer. 10.The synthetic microparticle of claim 9, wherein said at least onepolymer comprises said lactic acid monomer and said glycolic acidmonomer in a predetermined ratio.
 11. The synthetic microparticle ofclaim 10, wherein said predetermined ratio is 50:50.
 12. The syntheticmicroparticle of claim 10, wherein said predetermined ratio isdetermined by a mathematical model.
 13. The synthetic microparticle ofclaim 1, wherein the molecular weight of said at least one polymer isselected from the group consisting of approximately 10 kDa, a range ofapproximately 18-30 kDa and a range of approximately 40-78 kDa.
 14. Thesynthetic microparticle of claim 1, wherein the average molecular weightof said at least one polymer is approximately 15 kilodaltons.
 15. Thesynthetic microparticle of claim 1, wherein said microparticle isapproximately ten to twenty microns in diameter.
 16. The syntheticmicroparticle of claim 1, wherein said compartments range betweenapproximately 300-500 nanometers in diameter.
 17. The syntheticmicroparticle of claim 1, further comprising a pharmaceuticalformulation.